ArticlePDF AvailableLiterature Review

Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders

May 2021
Veterinary World 14(5):1220-1233

May 2021
14(5):1220-1233

DOI:10.14202/vetworld.2021.1220-1233

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Authors:

Ghadeer Sabah Bustani

University of Baghdad

Falah Hasan Baiee

University Of Kufa

Reproduction is fundamental for all living things as it ensures the continued existence of a species and an improved economy in animal husbandry. Reproduction has developed since history, and diverse processes, such as artificial insemination and in vitro fertilization, have been developed. Semen extenders were discovered and developed to protect sperm from harmful factors, such as freeze and osmotic shock, oxidative stress, and cell injury by ice crystals. Semen extenders preserve sperm by stabilizing its properties, including sperm morphology, motility, and viability and membrane, acrosomal, and DNA integrity. Therefore, semen extenders must provide a favorable pH, adenosine triphosphate, anti-cooling and antifreeze shock, and antioxidant activity to improve semen quality for fertilization. Hence, this review provides precise data on different semen extenders, preservative mechanisms, and essential additives for semen extenders in different animals.

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Veterinary World, EISSN: 2231-0916

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REVIEW ARTICLE

Open Access

Semen extenders: An evaluative overview of preservative mechanisms

of semen and semen extenders

Ghadeer Sabah Bustani1 and Falah Hasan Baiee2

1. College of Dentistry, The Islamic University, Najaf, Iraq; 2. Department of Clinical Science, Faculty of Veterinary

Medicine, University of Kufa, Kufa 54003, Najaf, Iraq.

Corresponding author: Ghadeer Sabah Bustani, e-mail: bustani@iunajaf.edu.iq

Co-author: FHB: falahhali@uokufa.edu.iq

Received: 19-12-2020, Accepted: 25-03-2021, Published online: 20-05-2021

doi: www.doi.org/10.14202/vetworld.2021.1220-1233 How to cite this article: Bustani GS, Baiee FH (2021) Semen

extenders: An evaluative overview of preservative mechanisms of semen and semen extenders, Veterinary World,

14(5): 1220-1233.

Abstract

Reproduction is fundamental for all living things as it ensures the continued existence of a species and an improved economy

in animal husbandry. Reproduction has developed since history, and diverse processes, such as artificial insemination and

in vitro fertilization, have been developed. Semen extenders were discovered and developed to protect sperm from harmful

factors, such as freeze and osmotic shock, oxidative stress, and cell injury by ice crystals. Semen extenders preserve sperm

by stabilizing its properties, including sperm morphology, motility, and viability and membrane, acrosomal, and DNA

integrity. Therefore, semen extenders must provide a favorable pH, adenosine triphosphate, anti-cooling and anti-freeze

shock, and antioxidant activity to improve semen quality for fertilization. Hence, this review provides precise data on

different semen extenders, preservative mechanisms, and essential additives for semen extenders in different animals.

Keywords: additives to semen extenders, artificial insemination, cryopreservation, egg yolk-based extenders, lipid

peroxidation, semen extenders.

Introduction

Artificial insemination (AI) is a powerful and

unique technique for fertilizing the females of most

mammals. The first animal was conceived using AI in

1784, and the first trial to produce straw for AI was at

the beginning of the 20th century [1]. AI was intended

to increase the number of insemination doses from a

single ejaculate but was unsuccessful because of the

absence of sperm-washing procedures for both chilled

or frozen–thawed semen of the male donor. These

techniques are considered the foundation stone in the

history of AI [1]. Moreover, AI is widely applied in

cattle [2] and humans [3] but less in sheep [4], buffa-

los [5], horses [6], deer [7], and other mammals [8].

To improve economic efficiency, AI can impact dif-

ferent productivity projects, such as cattle and sheep

husbandry and increasing weaning weight. Therefore,

AI has a remarkable ability to impact economic feasi-

bility [9].

Furthermore, AI needs fresh or well-preserved

semen, and 95% of all AI is accomplished using pre-

served semen [10]. Thus, semen must be preserved

in a perfect medium to maintain its quality [10-12].

Accordingly, it is necessary to develop and evaluate

semen extenders used to preserve semen during chill-

ing or cryopreservation [13].

This review provides precise data on semen

extenders, semen preservation mechanisms, and

essential additives for semen extenders in different

animals.

Preservation of Semen

The two primary techniques for semen storage

are chilling and cryopreservation. For the chilling

technique, semen is stored at 4-5°C for 3 days for

maximum and best results. In the cryopreservation

technique, semen is exposed to freezing for 3 h at 4°C.

Meanwhile, it is filled into 0.25-mL straws and finally

preserved and stored in liquid nitrogen for years [14].

Therefore, the crucial factors for long-term semen

preservation to retain its quality include cooling for

2-3 h, adding a cryoprotectant, and freezing in liquid

nitrogen [15].

Cooling Temperature for Semen Preservation

Temperature is decreased to induce sperm inac-

tivity during storage in liquid form. The globally

accepted cooling temperature is between 4°C and 5°C

to maintain semen quality and reduce gamete metabo-

lism in dogs[16], stallions [17], bulls [18], goats [19],

and rams [20], whereas in boars, the diluted liquid

semen must be kept between 15°C and 17°C [21].

Mammalian sperm cells, especially for rams [22],

boars [23], bulls [24], stallions [12], and men [25], are

damaged by rapid temperature drop. The temperature

of post-chilled semen may not be as harmful to the

intactness of the male sperm as the post-thawed tech-

nique, which results in low spermatozoal value [26].

A crucial advantage linked to using chilled semen

is the high fertility percentage compared with the

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freeze-thawed semen, which decreases the insem-

ination dose and increases the dose number, there-

fore, reducing storage expenses and easing the use of

AI [27]. Moreover, the lifespan of chilled semen in the

female reproductive tract is longer than that of frozen

semen and with a higher fertilization rate [28]. Singh

et al. [29] found that soybean-based diluent at 25%

gave good quality values for bull semen at 5°C at dif-

ferent time intervals. Studies showed that the supple-

mentation of different soybean lecithinconcentrations

in Tris-based semen extenders (containing 2% virgin

coconut oil) increased the functional parameters of

post- chilled bull semen [30].

Cryopreservation of Semen

Cryopreservation is the freezing of sperm, a

technique used to keep cells and tissues in a vital

state at −196°C in liquid nitrogen. The use of liquid

nitrogen started in the modern cryobiology era [31].

Furthermore, other advantages of cryopreservation

include the long-distance transportation of valuable

genetic materials and preventing the spread of patho-

gens [32].

Cryopreserved bull semen has been used com-

mercially in cattle for decades since the data illus-

trated that the conception rate of the cryopreserved

semen using AI technique is acceptable compared with

natural mating [33,34]. Thus, a single dose of cryopre-

served semen can achieve an equivalent in vivo fertil-

ization 8 times more than that of fresh semen [35,36].

Semen cryopreservation generates free radicals due

toexposure to atmospheric oxygen, and removes sem-

inal plasma from the sperm cells. This results in the

production of lipid peroxidation from sperm cells and,

consequently, increases in reactive oxygen species

(ROS) formation [37]. Furthermore, semen cryopres-

ervation has no standard protocol applied in most pre-

vious studies [38,39].

The improvement in semen extenders and cryo-

preservation techniques has significantly reduced the

harmful effects of cryopreservation. However, cryo-

preservation still causes sperm damage in humans and

various animals [40,41].

Factors that Determine Sperm Cryo-survival

The challenge during cryopreservation is not

sperm cells’ ability to resist the storage period in liq-

uid nitrogen but crossing the intermediate temperature

zone between −15°C and −60°C. The cells must go

through these temperatures twice, first during cool-

ing and then thawing, causing injurious effects on the

integrity of the sperm plasma membrane, acrosome,

and nucleus; mitochondrial function; and sperm

motility [32,42].

Cryopreservation techniques

Different cryopreservation techniques include

conventional freezing, directional freezing, and sperm

vitrification [43].

Conventional freezing techniques

Calling star-shaped ice crystal freezing is used in

the manual freezing technique [43], where the semen

is placed in contact with liquid nitrogen vapor at a

height 4-5 cm above for ≤15-10 min before storage

in liquid nitrogen. The freezing rate is approximately

60°C/min [15,43,44]. The conventional technique

varies among species depending on sperm quality or

cryo-survivability after freeze–thawing [44].

Directional freezing techniques

This technique is based on a multi-thermal gra-

dient approach [45,46]. After dilution with a freezing

extender, semen is chilled to 4-5°C at 0.3°C/min and

packaged in prechilled hollow tubes (5, 8, or 12 mL).

Tubes are advanced through a linear temperature gra-

dient, from 5°C to −50°C at a constant velocity of

1 mm/s. Then, seeding is done for 60 s through a cold

block at its other end, and tubes are moved to a collec-

tion chamber (−100°C). These tubes are collected and

transferred into liquid nitrogen for storage [43,47].

Sperm vitrification techniques

Vitrification is a process of transforming a solu-

tion containing a high concentration of cryoprotectant

in a glass-like state without ice crystal formation

through an ultra-rapid cooling process [48]. The high

cooling rate, high viscosity, and low volume enable

vitrification for sperm preservation [49]. In humans,

vitrified sperm exhibits high post‐thaw quality. Hence,

vitrification is introduced by solidifying the solution

into a glassy state without causing any crystallization

in a fast and inexpensive manner. Holding solution

is a significant component for vitrification prepared

with N-2-hydroxyethylpiperazine-N-2-ethane sul-

fonic acid -buffered Medium 199 and 20% of calf bull

serum [50].

Cryoprotectants Substance

The various anti-freezing cryoprotectants are

dimethyl sulfoxide (DMSO) and propylene glycol.

During freezing and thawing of isolated cells, mostly

5-15% cryoprotectant concentration must be main-

tained for better results [51].

Development of semen extender

Experts have developed media for sperm sur-

vival during the cooling and freezing techniques [15].

Many challenges, such as media toxicity [52], irreg-

ular pH [53], ROS [54], energy source [55], sperm

membrane damage [24], and cryo-shock preser-

vatives [56], were faced and have been resolved.

Extenders protect sperm, conserve motility, and fer-

tility over time by stabilizing the plasmalemma, pro-

vide energy substrates, and prevent harmful effects

of pH and osmolarity changes [57]. These solutions

facilitate the increase in fertilization rate using

high-quality extenders during chilling and cryo-

preservation. Therefore, a better quality of semen

extenders and additives should be added to increase

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semen quality and increase the rate of sperm fertil-

ization [15,58].

Semen extender

Semen extenders are used as a medium for pre-

serving sperm to enable fertilization. Semen extend-

ers can also maintain and preserve sperm metabolic

processes, control the pH of the medium during and

after post-thawing, control bacterial transmission and

contamination, and reduce cryogenic damage [10,59].

Likewise, semen extenders must provide other char-

acteristics, such as maintaining the pH at 6.8-7.2 [53],

provide energy [60], antioxidant to reduce the oxi-

dative stress [61], antibiotics to prevent contami-

nation [8], and anti-freezing shock [62,63]. These

properties keep the sperm storage and transport and

enable it to be used in AI in vitro fertilization, intra-

cytoplasmic sperm injection, and research stud-

ies. Extenders appear in two forms: Chilled (liquid

form) for an average of 3 days and cryopreserved for

years [64]. At present, several extenders use differ-

ent material sources such as animal source, egg yolk,

skimmed milk [65], and plant source (soybean leci-

thin) [24], which provide various features and diverse

problems according to the type of sperm extender

and species. A good trait of soybean lecithin semen

extender compared with the egg yolk extender is its

more hygienic nature [66]. Egg yolk semen extenders

are extensively used in the laboratory and field tech-

niques because of their reasonable price and satisfac-

tory results [24].

Various Components of Semen Extender

Non-penetrating cryoprotectant source

Skim milk and egg yolk are widely used

as non-penetrating cryoprotectants for preserv-

ing sperm [67] of different male mammals, such as

rams [68], bulls [59], stallions [65,69], boars [70],

and humans [29]. Because these extenders might pro-

tect sperm membranes, their acrosome and DNA may

be damaged because of high lecithin content [71].

Furthermore, soybean lecithin extenders can substitute

the animal source as a source of lipid/lipoprotein [72].

Egg Yolk

The first professor who used egg yolk in semen

extender was Philips in 1939 [73,74]. Egg yolk is the

primary non-penetrate substance used in extenders

to dilute semen and protect sperm from freeze shock

during the chilling process. Egg yolk-based extender

is commonly used in chilled, frozen semen, or both. It

works as a reservoir of cholesterol and phospholipids

that help protect the sperm cell membrane and acro-

some against cryogenic injury. Furthermore, it prevents

the loss of membrane phospholipids during the freez-

ing process [24,75]. Egg yolk protein has hydropho-

bic properties, which cannot penetrate the cell wall of

sperm. The low-density lipoprotein (LDL) of egg yolk

maintains sperm membrane phospholipids throughout

the cryopreservation processes [24,76]. The previous

works have shown that sperm are protected during

freezing by sequestering lipid-binding proteins from

LDL in the egg yolk [10,77,78]. It is also considered a

source of long-chain polyunsaturated fatty acids [79].

Besides, egg yolk contains lipid, protein and carbohy-

drate; it also contains minerals [10].

In contrast, several drawbacks against egg yolk-

based semen extender use include the wide range of

variability in the composition of egg yolk, the risk of

disease transmission or bacterial contamination, and

involvement of egg yolk in the microscopic exam-

ination of semen [39]. In general, egg yolk is used in

semen extenders at different concentrations. However,

it was also used at 20% (v/v). Pieces of evidence

revealed that LDLs are the egg yolk active ingredients

responsible for sperm protection [39,62].

Two types of extenders can be prepared from egg

yolk:

Egg yolk-based extender

Here, the concentration of the egg yolk is

20% [24,62,80]. Tris-buffered egg yolk extenders con-

taining fructose and glycerol preserve the fertility of

animal sperm at high extension rates. Poultry egg yolk

can be used in the semen extenders; however, some

studies show the possibility to use different egg yolks

of animals, such as quail [81], turkey [82], duck [83],

pigeon [82], goose [84], and ostrich [85]. Furthermore,

researchers found that quail’s egg yolk was improved

the quality of semen higher than other birds egg yolk

in terms of sperm motility and membrane integrity in

ram [86] and bull [82] semen. Nonetheless, Swelum

et al. [87] found that chicken egg yolk extenders are

most recommended for buck semen.

LDL extender

LDL (w/v) was prepared in the laboratory fol-

lowing the method described by Moussa et al. [88].

They used poultry egg yolks to isolate LDL by ultra-

centrifugation [22,88]. Higher kinetic parameters

were achieved using 2%, 4%, and 8% LDL compared

with 20% whole egg yolk in a Tris-milk extender and

can lower the concentration of LDL, such as 2% asso-

ciated with skimmed milk, which can be used for buf-

falo semen freezing [89].

According to several studies, egg yolk can be

used with a 20% extender concentration [24,82,90].

In contrast, LDL can be used with 8% concentra-

tion [91,92], and 2% LDL can be added solely with

skimmed milk [10,89]. One significant challenge of

using egg yolk and its derivatives is microbial con-

tamination by Escherichia coli. Consequently, the

fertilization capacity of contaminated semen could

negatively affect the risk of microbial contamination

associated with the egg yolk extender [93].

Milk Sources

Milk has been adapted for freezing mammalian

semen mostly in a reconstituted form combined with

arabinose, fructose, or egg yolk [10,44]. Skim milk

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proteins buffer semen pH and may also chelate any

heavy metal ions [94]. An important milk compound

is lactose, which is hydrophilic [95] and cannot dif-

fuse the cell wall of the sperm cells, which protects

the cell wall and prevents freeze shock [94-96]. Skim

milk-based extender is superior to Tris-based extender

based on semen preservation [96].

Soybean Lecithin

Soybean lecithin is an alternative for egg yolk and

has been developed and used commercially for semen

preservation [97]. Lecithin from soybean has been

successfully used for semen cryopreservation [98].

Nowadays, researchers believe that extenders free

from animal ingredients can decrease the risk of con-

tamination transported by the animal source [93,99].

Therefore, soybean lecithin can be used as an alterna-

tive to milk- or egg yolk-based extenders for semen

cryopreservation in bulls [24,30,98], boars [100],

rams [101], dogs [102], deer [103], and camels [104].

The active components of soybean lecithin and

egg yolk are entirely identical. These components are

oleic acid, palmitic acid, stearic acid, and phospha-

tidylcholine. They prevent the diluted semen from

freeze shock. The prevailing phospholipids in most

mammalian biological membranes can confer phys-

ical stability to sperm cells [105]. On the basis of

the research results of Gamal et al. [97] and Miguel-

Jimenez et al. [98], soybean lecithin-containing

diluter is possibly the best semen extender in bulls.

Furthermore, El-Keraby et al. [106] found that the use

of soybean-based extender increases sperm functional

motility and reduces bacterial contamination in freeze–

thawed bull semen. In a similar vein, Zhang et al. [107]

ensured that the semen extender supplemented with

soybean lecithin at 6% could upgrade sperm general

and progressive motility and intact plasma membrane

of post-thawed male boar sperm cells. Conversely,

Singh et al. [26] and Rehman [71] discovered that 25%

soybean-based extender could improve sperm motility,

sperm viability, sperm membrane integrity, and acro-

somal integrity of bull sperm at 5°C.

Depending on the results of Fathi et al. [108],

using Tris-soybean lecithin-based extender at a 3%

concentration can be an appropriate alternative to

either BullXcell® or OptiXcell® in Damascus goat

sperm cryopreservation.

Glycerol as an Anti-shock

After discovering glycerol as a remarkable cryo-

protective agent for cryopreserving semen, using

liquid nitrogen for adequate storage of frozen semen

and AI has been a valuable and prevalent repro-

ductive biotechnology for cattle genetic improve-

ment [24]. Discovering the cryoprotective properties

of glycerol in 1949 enabled the cryopreservation of

different animals’ sperm . [109]. Although different

cryoprotective substances have been tested, including

DMSO and propanediol, glycerol remains the favorite

cryoprotectant for semen cryopreservation. Glycerol

is a dominant cryoprotectant that can cross the cell

membrane [101]. Studies stated that glycerol could

be added to the semen at different temperatures. For

instance, Evans et al. [110] observed that good semen

protection was achieved at 30°C. In contrast, another

suggested that procedure for semen cryopreservation

is adding glycerol at 5°C [14,111]. Furthermore, stud-

ies also suggested that freezing extenders containing

3% glycerol combined with the straw freezing method

using dry ice can produce the best post-thaw quality

parameters for boar semen [112].

Fernández et al. [113] revealed that semen

extenders containing egg yolk with 6% glycerol, fol-

lowed by a rapid cooling rate, could yield higher post-

thaw outcomes for epididymal sperm compared with

semen extenders containing 3% glycerol. In another

study, Martínez et al. [114] demonstrated that extend-

ers containing 4% glycerol with 10% egg yolk are

most suitable.

DMSO is a cryoprotectant that quickly enters

sperm cells. It can be used to maintain the frozen

sperm quality of bulls [115], boars [29], goats [116],

and dogs [117]. In stallions, it is preferable to use

DMSO or methyl formamide alone or combined with

glycerol in skim milk extenders as cryoprotectants

and alternatives to extenders containing glycerol

only [118,119]. A study that compared three cryo-

protectants, glycerol, ethylene glycol, and DMSO,

revealed that the post-thaw motility and fertilization

capacity of glycerol were higher than those of eth-

ylene glycol and DMSO in dogs [120]. Earlier studies

revealed that ethylene glycol is more dominantly used

in buffalos [87], bulls [121], and sheep [122].

Source of Energy

Energy intake is responsible for the continuation

of development and the function of all living cells, and

gametes are no exception. Two metabolic pathways

are producing adenosine triphosphate (ATP), that sup-

plies energy for the main functions of sperm, which

are oxidative phosphorylation and glycolysis [123].

Glycolysis occurs in the cytoplasm of sperm cells and

provides energy for sperm metabolism [123]. Sugar,

such as fructose and glucose, is considered the pri-

mary energy source in sperm cells [124]. However,

fructose is the best sugar for maintaining functional

membrane integrity, adequate sperm motility, and-

tonic after thawing [125].

Disaccharides are considered non-permeating

agents for cells. These sugars interact with phospho-

lipids of the plasma membrane, increasing sperm sur-

vival post-cryopreservation [126]. Moreover, lactate

and pyruvate are significant energy sources in stal-

lion sperm with dose effects on mitochondrial func-

tion, motility, and ROS production [127]. Trehalose

can be used as a cryoprotectant in semen extenders to

preserve the optimal quality of motility, viability, and

membrane integrity of goat sperm cells compared with

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other types of sugar [128]. Glucose is a component

of egg yolk and, therefore, can be used as an energy

source. Nonetheless, other kinds of sugars, such as

galactose, sucrose, maltose, xylose, and raffinose, have

been successfully used for frozen bull semen [10,11].

Various Components as Additives to Semen

Extenders

To promote the quality of the extenders, sev-

eral studies have been undertaken to use different

materials and compounds such as those of plant ori-

gins [24,129-131], whole milk [54], fish oil [132], and

honey [133], which contain natural compounds such

as antioxidants [24,130,131].

Added Components of Plant Origin

There is an international demand for using natural

medical sources in semen extenders of different ani-

mals [134,135], for example, strawberry [136], green

tea [134], virgin coconut oil [30], pomegranate [135],

and Pinus brutia [137,138], among others. The effects

of several plant extracts on fertility have been demon-

strated as antioxidants in many animal species due to

their free radical scavenging properties [139].

According to the results of El-Sheshtawy [136],

he used a 1%–5% concentration of strawberry in Tris

extender and improved semen parameters in cooling

temperature and used strawberry at 3-6% concentra-

tion, which improved semen parameters in freezing

temperature for bull semen.

Researchers found that supplementation of Tris-

citric acid extender with 1.0% green tea improved

sperm parameters in both in vitro and in vivo fertil-

ization, which decreased lipid peroxidation in buffalo

bull sperm freezing and thawing processes [134].

For virgin oil addition, researchers found that

Tris-based extenders containing 2% virgin oil did not

improve the quality of parameters for freeze–thawed

bull semen but enhanced the quality of parameters for

chilled bull semen [30]

An experimental study illustrated that adding P.

brutia to the semen extender does not improve param-

eters such as motility but prevents chromatin damage

and reduces oxidative stress and sperm abnormalities

when used at a concentration of 50 μg/mL [137].

Added components of animal origin

Honey

Malik [133] found that adding honey to extend-

ers significantly affected sperm motility before freez-

ing and sperm abnormality of the freeze–thawed

semen. Honey contains a high number of various sim-

ple sugars and antioxidants [140]. Furthermore, honey

is also a highly concentrated product. It has a poten-

tial hyperosmotic extracellular environment around

sperm cells that enhance the efflux of intracellular

fluid, thereby minimizing the formation of ice crystals

inside the sperm cytoplasm, which has been linked to

sperm damage during cryopreservation [141]. This

illustrated that using honey increased the quality of

the semen after thawingcompared with using egg yolk

extenders.

Yimer et al. [142] showed that adding honey

to bull semen Tris extender at 2.5% was optimum

to obtain improved semen cryopreservation results

compared with Bioxcell®. Another experiment [143]

illustrated that adding d ifferent honey concentrations

to extenders has various effects on the semen quality

of different bull breeds. Jersey bull exhibited the best

sperm quality compared with Mafriwal, Piedmontese,

and Limousin bulls. Because El-Nattat et al. [144]

considered that 1% concentration of honey additive to

Bioxcell® extender could be higher effectively when

used in bull semen cryopreservation compared with

Bioxcell® only identified as a control.

Moreover, Fakhrildin and Alsaadi [141] showed

that adding of honey to the freezing semen medium

of humans at 10% concentration to semen extender

resulted in enhanced sperm post-thawing quality of

most sperm parameters.

In addition, researchers have reported the benefits

of using honey as a supplement in the cryopreservation

semen media of various animals, such as goat [145],

which act as natural antibiotics against pathogenic

bacteria, hinder sperm survival, fertilizing ability,

reduce the number of dead abnormal sperm, and acro-

somal damage. Some studies showed that using 2.5%

honey might be an energy source to ram semen [146].

El-Sheshtawy et al. [147] illustrated that adding 3% of

honey in extenders as a cryoprotectant improved Arab

stallion post-thawing sperm parameters.

Fish oil

Fish oil can improve semen performance after

freeze–thawing and AI besides the type of extender

shown in bulls. The addition of 150 mg/100 mL fish

oil in the extender could positively enhance the quality

of post-thawed semen of Kalang swamp buffalos [59].

Abdi-Benemar et al. [148] found that adding 0.30 g of

fish oil per 100 mL of egg yolk-based extender resulted

in an improved fertility capacity of ram and goat semen.

Kaeoket et al. [149] found that adding fish oil to boar

semen extender improves sperm motility, viability, and

acrosomal integrity. The researcher also showed that

fish oil supplementation has a beneficial effect on semen

quality, except for in vitro evaluations [150].

Some studies reported that the addition of fish oil

to feed supplements improved the semen quality and

fertility rate of sheep [151], bulls [152], rams [153],

boars [154], and stallions [155].

Vitamins

Vitamins are added to extenders to improve

semen function parameters for liquid nitrogen storage

or cryopreserved sperm cells because vitamins are

non-enzymatic antioxidants [156-159].

Vitamin B12

Adding Vitamin B12 to the extenders improved

bull frozen semen quality, elevated the motility

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percentage of sperm cells, and improved movement

characteristics [68,86,160,161]. Researchers found

that the addition of 2.50 mg/mL Vitamin B to semen

extenders improved bull frozen semen parameters

and quality [161]. Furthermore, oral administration of

200 mg/kg body weight per day of Vitamin B12 could

improve fresh and post-thawed sperm quality and fer-

tility in male broiler breeders [159].

Vitamin E

Vitamin E is a cellular stabilizer of unsaturated

lipids against oxidative deterioration, and hence, it

maintains the structural and functional integrity at the

subcellular level [161,162]. In general, Vitamin E is

the primary component of the antioxidant system in

sperm cells [163,164]. Furthermore, adding Vitamin

E to Tris-egg yolk extenders at 60 and 120 μM pro-

vides higher integrity to the plasma membrane,

mitochondria, and kinematic parameters of sperm

cells of rams [165], roosters [162], and bulls [164]

post-cryopreservation.

Vitamin C

Vitamin C is the most crucial antioxidant in

seminal fluid [166]. Researchers found that 0.9 mg/

mL of Vitamin C improves the longevity and quality

of chilled sperm in Awassi ram semen stored at 5°C.

Furthermore, as an alternative to glutathione, Vitamin

C is considered more efficient in protecting ram

sperm viability and acrosomal integrity than Vitamin

E because Vitamin c can neutralize H2O2 production

in a hydrophilic environment [167,168] by preventing

peroxide formation [169]. However, higher concen-

trations of Vitamin C (2.5 mM) proved to be harmful

to sperm motility in freeze–thawed bull semen [168].

Studies in humans showed that a high concentration

of Vitamin C in the range of 0.02-0.6 mM adversely

affected sperm motility [170].

Other Additions

Several researchers have added various sub-

stances to semen extenders, such as milk, caseinate,

and lactoferrin [120]. Other researchers added hor-

mones, such as insulin [171], follicle-stimulating

hormone [172], and testosterone [173], to the semen

extenders. The addition of selenium improved male

reproductive performance by potentiating semen qual-

ity and suppressing free radicals [174,175]. Selenium

could decrease lipid peroxidation and increase anti-

oxidants in rooster seminal plasma after the freeze–

thawing process [176].

Antibiotic addition to semen extenders

Antibiotics are added to semen extenders to

reduce microbial contamination of the external envi-

ronment or during semen collection. Different antibi-

otics, such as penicillin and streptomycin, ceftiofur,

apramycin, and aminoglycosides or linco-spectin

+ tylosin + gentamycin, have been added to semen

extenders [10].

Commercial extenders

Several commercial extenders are used for

diluting and preserving semen during cooling and

cryoprotection. Optidyl® and Triladyl® (Biovet,

France) are commercial extenders containing egg

yolk and provide excellent protection for bull semen

against freeze shock. It is usually used by many

French AI centers [76]. Bioxcell® is a commercial

extender that contains milk, egg yolk, or both [177].

Gent® A (Minitüb GmbH, Tiefenbach, Germany)

is a commercial extender containing egg yolk and

is used for the preservation of semen for a long

time [178]. BotuSemen® is a commercial extender

that contains a skim milk base used for preserving

frozen sperm of stallions [12]. EquiPlus® is a com-

mercial extender that contains defined milk proteins

used to preserve semen [26]. INRA 96® is a com-

mercial extender that contains a caseinate used to

preserve semen [179].

Semen Assessment

Sperm motility

The motility of sperm is the most critical param-

eter for evaluating male potential fertility [77], and it

depends on mitochondrial function [180,181]. Motility

includes total general motility, progressive motility,

and kinematic parameters [181]. Sperm cells must

possess high maintenance of sperm motility to ensure

maximum potential fertility [114,182]. Moreover, the

motility of sperm cells is also associated with sperm

DNA defects. Furthermore, sperm motility is crucial

for fertilization [45,181]. Immotile sperm and motil-

ity disorders of sperm are notable indicators of male

infertility [183]. Therefore, microscopic examination

and estimation of the percentage of forward move-

ment of sperm are a standard test to indicate male fer-

tility [45,181].

The tail of mammalian sperm cells is represented

by a single, specific type of motile cilium known as

the sperm tail (flagellum) that generates its movement

to propel the cell through the female reproductive tract

and fertilize the oocyte [184]. Sperm cells rely on vig-

orous motility that is initiated once they are released.

They reach capacitation, which is needed to hyper-

activate the ability to perform the acrosome reaction

and fertilization [161,162]. In general, the tail motion

is generated by ATP. When activated, sperm cells

exhibit vigorous motility and enter rapid consumption

of intracellular energy in which ATP content could be

relevant for fertilizing potential [185].

Computer-assisted sperm analysis has been used

to collect data after reducing human inequality during

semen estimation. Moreover, it could be noted that

different sperm motilities deem a fundamental part of

semen evaluation in almost all creatures [186]. The

most critical kinetic parameters include curve–lin-

ear velocity, straight-line velocity, and average-path

velocity [182].

Veterinary World, EISSN: 2231-0916 1226

Available at www.veterinaryworld.org/Vol.14/May-2021/21.pdf

Sperm viability

The viability of sperm cells is an essential aspect

of ejaculation quality that determines competitive fer-

tilization success, proportional to live sperm [187].

Spermatozoal viability is essential for motility and fer-

tilizing ability. Once sperm viability is reduced, their

ability to induce fertilization is decreased [58,187].

The percentage of live sperm cells was determined in

the laboratory by identifying the number of sperms that

did not take up the eosin–nigrosin stain [14,58,187].

Sperm morphology

Sperm morphology depends on spermiogene-

sis [188] or events that occur after spermiation [175].

Improper handling of semen samples because of lack

of experience can cause challenges during the cooling

and freezing processes. Furthermore, these challenges

may lead to acrosomal damage and abnormality of

sperm tails [189]. Sperm shape abnormality includes

head defects, such as microcephaly and macroceph-

aly. Midpiece defects, such as proximal cytoplasmic

droplets, distal midpiece reflexes, and segmental apla-

sia of the mitochondrial sheath, are common [190].

There are three theories about abnormal sperm

that can be presumptive. The first theory suggests pri-

mary or secondary defects; the primary defect occurs

at the seminiferous tubules in the progress of spermato-

genesis. In contrast, the secondary sperm abnormality

is caused by abnormal function of the epididymis or

during semen handling after ejaculation [139]. The

second theory ascribes the sperm defect to the rela-

tionship between sperm and the fertility of males.

Therefore, it is defined as a major or minor defect. The

major sperm defect can directly affect male fertility;

however, the minor defect may not affect male fertil-

ity [191]. Whether the defects of sperm cells are com-

pensable or un-compensable, it distinguishes between

the types of sperm defect. This may be compensated

for by increasing the numbers of sperm to overcome

sperm defects [192]. The third theory states that sperm

defects can be classified according to the defect site

(head, midpiece, and tail). Sperm morphology can be

evaluated either with non-stained wet models under

phase-contrast microscopy orfixed and stained sperm

cells with post-dried eosin–nigrosine stain under 100×

magnification [193].

Acrosomal Reaction

The study of acrosomal integrity in mammalian

species is a valuable tool in evaluating male subfer-

tility and infertility. With advancements in micro-

scopic visualization and cell-staining technology,

methods for determining acrosomal integrity have

been developed. The acrosome reaction is an exo-

cytotic event initiated when sperm binds to the zona

pellucida of an ovum. Once sperm binding occurs,

the outer acrosomal membrane fuses with the overly-

ing plasma membrane of the ovum [194]. The fusion

of these membranes triggers vesiculation, a process

where many small vesicles are created that allows for

the dispersal of acrosomal enzymes [195,196]. The

release of acrosomal enzymes enables the sperm cell

to digest its way through the zona pellucida and begin

the process of fertilization [197,198].

Importance of an Intact Acrosome

The ability of a sperm cell to undergo capaci-

tation, acrosome reaction, and a fertilization event

requires an intact acrosome at the time of ejaculation

and after the freeze-thawing process. The disruption

or damage to the acrosome is permanent and results in

premature loss of acrosomal contents, ultimately pre-

venting fertilization [199,200]. Moreover, damaged

acrosomes do not undergo vesiculation properly but

spontaneously rupture and defect fertilization in the

end [199].

Hypo-osmotic Swelling Test (HOST)

The HOST evaluates the functional integrity of

the sperm’s plasma membranes. Although the HOST

is a simple test, it is considered an indicator of fertility

in some species as the viability of the sperm membrane

is an essential requirement for fertilization [50,201].

HOST and the integrity of the acrosome could be

associated with motility results. To evaluate the sperm

membrane integrity using HOST, an aqueous solu-

tion is prepared with fructose and trisodium citrate in

distilled water to produce a solution (100 mOsm/kg

H20) as described by Lamia et al. [202] and Kumar

et al. [99].

In vivo Fertility Evaluation

It is crucial to examine the in vivo fertility test

to provide comprehensive and perfect data. This test

is considered the most important one for evaluating

semen quality and provides information on the capa-

bility of sperm and pollination capacity of the ova

after access to the female reproductive tract. Potential

fertility depends on multiple parameters that require

a multi-parametric analysis of sperm morphology,

sperm motility, membrane status of sperm, sperm

acrosome reaction, and genome integrity of sperm to

provide a complete picture of a male’s fertility poten-

tial. Moreover, the reliability of fertility prediction

is reported to increase by combining several in vitro

sperm quality parameters [180,181,203]. The diffi-

culty of this assay is that it is a time-consuming and

costly procedure as hundreds of successful insemina-

tions are required.

Lipid Peroxidation Test

A typical trait of biological cell membranes

is the asymmetrical organization of fatty acids (lip-

ids) within the bilayer. The composition of lipids in

most mammalian sperm cell membranes is different

compared with that in somatic cells. Polyunsaturated

fatty acids are found in high amounts in sperm cell

membranes, and the ratio of saturated to unsaturated

fatty acids in ruminants’ sperm cell membranes is

Veterinary World, EISSN: 2231-0916 1227

Available at www.veterinaryworld.org/Vol.14/May-2021/21.pdf

higher than in other animals. This makes sperm cell

membranes more subject to damage by peroxidation,

primarily when an ROS is present [37,204]. In con-

trast, controlling the release of molecular oxygen may

result in less ROS production and hence maintain the

fertilizing ability, acrosome reaction, and capacitation

of sperm [37,205].

Conclusion

Our review article provides a comprehensive

analysis of different semen extenders, the mechanisms

of preserving semen, and essential additives for semen

extenders in different animals. The study can be used

as a road map for future studies to develop appropriate

semen preservation and AI techniques.

Authors’ Contributions

FHB: Conceptualized, drafted, and supervised

the final version as well as editing of the review.

GSB: Collected relevant literature, contributed to the

original draft, data curation, investigation, and review

of the manuscript. All authors read and approved the

final manuscript.

Acknowledgments

We would like to thank Prof. Abbas H. J. Sultan,

for English editing. The authors did not receive any

funds for this study.

Competing Interests

The authors declare that they have no competing

interests.

Publisher’s Note

Veterinary World remains neutral with regard

to jurisdictional claims in published institutional

affiliation.

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The addition of duck egg yolk in skim milk – fructose extender maintains sperm motility and viability of Muscovy duck (Cairina moschata) at 5°C storage

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Full-text available

Mar 2024

This study aimed to determine the addition of duck egg yolk in skim milk – fructose extender on sperm motility and viability of Muscovy duck (Cairina moschata) at 5°C storage. Ejaculates of Muscovy duck were divided into four portions equally. The first portion was for the control group (diluted in skim milk – fructose extender), and the second, third, and fourth portions were for the treatment groups of T1, T2, and T3 (diluted in skim milk – fructose extender added with 2.5, 5.0, and 7.5% v/v duck egg yolk). Sperm motility and viability were evaluated two hourly until the motility percentage reached 40%. The data were analyzed using ANOVA and Duncan test. The result showed that the highest percentage of spermatozoa motility and viability (p<0.05) was in the T2 group compared to the other groups. It could be concluded that the Muscovy duck semen extended in skim milk - fructose with 2.5 to 7.5% egg yolk of duck qualified for artificial insemination at storage up to 10 hours in 5°C storage.

The effect of bee drone brood on the motility and viability of stallion spermatozoa—an in vitro study

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Full-text available

May 2024
IN VITRO CELL DEV-AN

Bee drone brood is a beehive by-product with high hormonal activity used in natural medicine to treat male infertility. The aim of the study was to assess the effect of drone brood on stallion spermatozoa during a short-term incubation for its potential use in the equine semen extenders. Three different forms of fixed drone brood (frozen (FR), freeze-dried (FD), and dried extract (DE)) were used. Solutions of drone brood were compared in terms of testosterone, protein, total phenolic content, and antioxidant activity. The stallion semen was diluted with prepared drone brood solutions. The computer-assisted semen analysis (CASA) method was employed to evaluate the movement characteristics of the diluted ejaculate. To determine spermatozoa viability, the mitochondrial toxicity test (MTT) and Alamar Blue test were performed. In terms of testosterone content and antioxidant activity, a close likeness between FR and FD was found whereas DE’s composition differed notably. FR had a positive effect mainly on progressive motility, but also on sperm distance and speed parameters after 2 and 3 h of incubation. On the contrary, FD and DE acted negatively, depending on increasing dose and time. For the first time, a positive dose-dependent effect of fixed drone brood on spermatozoa survival in vitro was demonstrated. Graphical Abstract

Effects of liquid storage of buck semen at refrigeration temperatures on sperm viability and fertility to develop ready to use goat semen diluent

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Jan 2024

It is generally acknowledged that lower pH throughout liquid storage can decrease sperm motility and energy production, which may be advantageous for maintaining sperm viability during cryopreservation. Hence, the purpose of this study was to see how pH affected sperm viability, motility, membrane integrity, and oxidative changes after 5 days of storage in chemically specified extenders at refrigerator temperatures to formulate a ready-to-use goat semen diluent. Ejaculates were extended with tris-citric acid-fructose diluent with 20% or without egg yolk and stored at 5°C for 5 days. Sperm motility, live count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, pH, and malondialdehyde all substantially differed significantly (P < 0.05) during different days of liquid storage. The pH of diluted semen decreased considerably (P < 0.05) from its original pH of 6.46 on 0 day to pH 6.06 on the 5th day of liquid storage at refrigeration temperature. Diluted semen at refrigeration temperature having 20% egg yolk had significantly higher (P < 0.05) seminal characteristics compared to liquid semen without egg yolk. The liquid storage of semen quality became worse day by day due to a drop in pH and egg yolks acted as a cryoprotectant against the harmful effects of chilling. Hence, 20% egg yolk may be used to enhance the keeping quality of diluted buck semen at refrigeration temperature for up to 5 days. Based on this result, we formulated a ready to use goat semen diluent for liquid semen storage at room temperature and refrigeration temperature.

Sperm Incubation in Biggers–Whitten–Whittingham Medium Induces Capacitation-Related Changes in the Lizard Sceloporus torquatus

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Full-text available

May 2024

Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that ensure reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. We aimed to perform a sperm analysis to determine if capacitation-related changes were induced after incubation with capacitating media. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 μL using BWW medium to incubate with 5% CO2 at 30 °C for a maximum duration of 3 h. A fraction was retrieved hourly for ongoing sperm assessment. The sperm analysis included assessments of its motility, viability, the capacitation status using the chlortetracycline (CTC) assay, and the acrosome integrity with the lectin binding assay to detect changes during incubation. We found that total motility was maintained up to 2 h post incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The CTC assay indicated a reduction in the percentage of sperm showing the full (F) pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. Our observations support the notion that this process may also occur in saurian. While sperm analysis is a valuable method for assessing certain functional changes, additional approaches are required to validate this process.

EFFECT OF SPERMS FREEZABILITY ON SOME SEMEN ATTRIBUTES AND AMINO ACID CONCENTRATIONS IN SEMINAL PLASMA OF HOLSTEIN BULLS

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Full-text available

Apr 2024

This study investigated the effect of different sperm freezability on some semen characteristics and amino acid concentrations in the seminal plasma of Holstein bulls cryopreserved for two years. The semen straws (0.25 ml) that cryopreserved for two years were divided into three equal groups (7 bulls per group) according to the sperm's freezability: medium (73.02%; F1), good (83.76%; F2), and high (91.20%; F3). The F1 bulls were superior (P≤ 0.05) in sperm's cell individual motility (SCIM) for fresh semen as compared with F2 and F3 bulls. The F2 and F3 groups were superior (P≤ 0.01) to the F1 group in live sperm percentage at cryopreservation. The sperm's DNA damage percentage was lesser (P≤ 0.05) in the F1 and F3 groups as compared with the F2 group. The differences among groups in normal sperm percentage, sperm's plasma membrane and acrosome integrity percentage, malondialdehyde, and total antioxidant concentrations lacked significance. The essential and non-essential amino acid concentrations in seminal plasma did not differ among groups. In conclusion, sperm freezability influenced some semen attributes but did not affect amino acid concentrations in the seminal plasma of Holstein bulls.

Effect of the addition of soursop juice (Annona muricata L., 1753) to egg yolk citrate extender on the quality of bull semen

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Full-text available

Apr 2024

The present study was carried out to investigate the effect of the addition of soursop (Annona muricata L., 1753) juice to the citrate-egg yolk extender (EYC) on the quality of cryopreserved bull semen. After pre-evaluation, the semen was distributed in five extenders T1, T2, T3, T4 and T5 containing respectively 0% soursop juice (SJ) (T1), 25% SJ (T2), 50% SJ (T3), 75% SJ (T4), 100% SJ (T5) and microscopic evaluation was performed daily to assess plasma membrane integrity (PMI), progressive motility of spermatozoa (MP) and sperm pH until all sperm were dead. The results indicate a significant increase in spermatozoa PMI and MP (p<0.05) in the case of treatment with 50% SJ (T3). Spermatozoa showed a motility of 50.78% 9 days later in the citrate-egg yolk extender containing 50% SJ (T3), whereas at day 9, spermatozoa in the other extenders had a progressive motility of less than 50%. Similar results were obtained for PMI with 50.78% of spermatozoa having an intact plasma membrane 9 days later in the citrate-egg yolk extender containing 50% SJ whereas the PMI of cells in the other stents was less than 50%. However, the pH of the extender containing 50% SJ was closest to 7 during the experimental period. The results of this experiment suggest that the addition of 50% soursop juice (T3) to the citrate-egg yolk extender could be adopted as an acceptable alternative to traditional egg yolk-based extenders for the cryopreservation of bull semen.

Effect of Different Natural Extenders in Maintaining the Quality of Sperm Fish (Cyprinus carpio)

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Full-text available

Feb 2024

High demand on as consume fish nor as decorative fish, causes an increasing production. But, an obstacle occurs on production, due to reproductive characteristic of Common Carp fish. Different time on sperm and eggs production from male and female fish cause difficulties in breeding. Hence, it needed sperm preservation so the breeding could be done in whole year. Natural extenders are the solution to preserve fish sperm without side effect. The purpose of this study was to determine the effect of different types of extenders and different doses on the percentage of sperm motility and viability of carp (Cyprinus carpio) after storage This research was conducted at the Faculty of Fisheries and Marine Sciences, Universitas Brawijaya and the Freshwater Cultivation Installation (IBAT Punten), Batu, East Java. The method in this study was Complete Random Design (CRD) with 6 treatments, K (100ml lactate ringer); A (1ml cider + 99ml lactate ringer); B (1ml coconut + 99ml lactate ringer); C (1ml date juice + 99ml lactate ringer); D (1ml honey + 99ml lactate ringer); E (1ml sugar cane + 99ml lactate ringer). The results showed that the highest motility was obtained in the treatment of using date extract extender with a motility percentage rate of 77.66%; highest viability was obtained in the honey extract with 80.71%; and the highest fertility rate was obtained in the honey extract treatment with 72.67 %.

The Effects of Different Antimicrobial Peptides (A-11 and AP19) on Isolated Bacteria from Fresh Boar Semen and Semen Quality during Storage at 18 °C

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Full-text available

May 2024

Antibiotic resistance (AMR) is a major public health concern. Antimicrobial peptides (AMPs) could be an alternative to conventional antibiotics. The purpose of this research was to investigate the antimicrobial ability of the synthetic AMPs (i.e., A-11 and AP19) on the most frequently isolated bacteria in boar semen and their effect on extended boar semen quality during storage. We tested the antimicrobial effect of A-11 and AP19 at different concentrations and compared them with gentamicin for inhibiting the growth of E. coli, Pseudomonas aeruginosa and Proteus mirabilis that were isolated from fresh boar semen. In order to evaluate the effect of AMP on semen qualities on days 0, 1, 3, and 5 after storage at 18 • C, seven fresh boar semen samples were collected, diluted with semen extender with antibiotic (i.e., gentamicin at 200 µg/mL, positive control) or without (negative control), and semen extender contained only A-11 or AP19 at different concentrations (i.e., 62.50, 31.25, and 15.625 µg/mL). The total bacterial count was also measured at 0, 24, 36, 48, and 72 h after storage. Comparable to gentamicin, both A-11 and AP19 inhibited the growth of E. coli, Pseudomonas aeruginosa, and Proteus mirabilis at 62.50, 31.25, and 15.625 µg/mL, respectively. Comparing the total bacterial count at 0, 24, 36, 48 and 72 h after storage, the lowest total bacterial concentration was found in the positive control group (p < 0.05), and an inferior total bacterial concentration was found in the treatment groups than in the negative control. On day 1, there is a lower percentage of all sperm parameters in the AP19 group at a concentration of 62.50 µg/mL compared with the other groups. On day 3, the highest percentage of all sperm parameters was found in the positive control and A-11 at a concentration of 31.25 µg/mL compared with the other groups. The AP19 group at 62.5 µg/mL constantly yielded inferior sperm parameters. On day 5, only A-11 at a concentration of 15.625 µg/mL showed a total motility higher than 70%, which is comparable to the positive control. A-11 and AP19 showed antimicrobial activity against E. coli, Pseudomonas aeruginosa and Proteus mirabilis isolated from boar semen. Considering their effect on semen quality during storage, these antimicrobial peptides are an alternative to conventional antibiotics used in boar semen extenders. Nevertheless, the utilization of these particular antimicrobial peptides relied on the concentration and duration of storage.

Sperm Incubation in Bww Medium Induces Capacitation- Related Changes in the Lizard Sceloporus torquatus

Preprint

Full-text available

Mar 2024

Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that facilitate reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 μl using BWW medium to incubate with 5% CO2 at 30 °C for a maximum duration of 3 hours. A fraction was retrieved hourly for ongoing sperm assessment. We found that total motility is maintained up to 2 hours post-incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The chlortetracycline assay indicated a reduction in the percentage of sperm showing the F pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. These observations support the notion that this process may also occur in saurian.

Non-Permeable Cryoprotectants Improves the Antioxidant Capacity and Viability of Frozen-Thawed Donkey Semen

Preprint

Jan 2024

Influence of adding different energy sources to the bull and ram spermatozoa exposed to different refrigerating times

Article

Full-text available

Aug 2019

Semen storage changes sperm synthesis and biochemical characteristics and hence hinders ferti-lizing ability. Mixture of two various sugar molecules appeared an improved positive impact on stored spermatozoa of different species. The current study aimed to investigate the effect of fructose, sucrose and raffinose supplementation to Tris-glucose-egg yolk extender (TGY) beside comparing their effect with the effect of commercial Triladyl® extender on bull’ and ram’ semen stored at 5 °C for 96 hours. The semen samples were pooled, extended and divided into five portions. TGY or Triladyl® extenders were diluted at rate 1 part of raw semen: 5 parts of the extender. The TGY diluent was split into four diluent parts in sterilized test tubes. TGY and Triladyl® extenders contained one type of sugar, while the other three extended semen samples contained two types of sugar including TGY plus 0.990 g fructose (TGYF), 0.700 g sucrose (TGYS) and 0.700 g raffinose (TGYR) /100ml extender. The ex-tended semen was stored at 5°C for 0, 24, 48, 72 and 96 hours. The obtained data show that spermato-zoa parameters significantly (P < 0.01) decreased with increasing preservation period of all diluent samples. Meantime, supplementation with fructose, sucrose or raffinose with TGY extended samples improved significantly (P < 0.01) sperm properties (motility, livability with acrosomal status and normality, enzymatic actions of AST, ALT, LDH, and ALP, and sperm penetration ability for both bull and ram diluted semen. Concerning the sperm characteristics of the commercial Triladyl® extend-er, it was better than those of TGY without any supplementation for both bull and ram diluted semen. In conclusion, the obtained results suggest that obtaining a reasonable and advantageous impact for the preservation of bull and ram spermatozoa could be acquired by using diluents containing a sugar blend. However, as these results depend on in vitro assessments, there is a need for further fertil-ity trials to confirm it. Keywords: Extended semen, sugars, preservation, and penetration.

Crioconservación seminal en peces de agua dulce: aspectos biotecnológicos, celulares y bioquímicos

Article

Full-text available

Nov 2020

La crioconservación es una herramienta biotecnológica que en peces está orientada principalmente a la conservación criogénica de semen como estrategia de preservación del recurso genético y a su uso para la producción de alevinos con fines diferentes. Actualmente, los protocolos de crioconservación seminal en peces de agua dulce establecen una amplia variedad de procedimientos cuya efectividad se basa en aspectos ligados a la calidad seminal post-descongelación y la fertilidad, así como su relación con el desarrollo de la progenie. El efecto de la conservación del semen en nitrógeno líquido por periodos amplios de tiempo también toma importancia en ésta biotecnología. Por lo anterior, el objetivo de la presente revisión es describir aspectos biotecnológicos, celulares y bioquímicos asociados al proceso de crioconservación seminal en peces dulceacuícolas, resaltando los avances, las limitaciones y sus perspectivas.

Calcium Administration to Improve Parturition in Dairy Cows

Article

Full-text available

Nov 2020

Parturition in bovine consider as a stressful event to the calving cows and calves as well. In general divided into three phases. The former involves the endocrinal and mechanical arrangement of uterine and cervical tissues, embryo and birth the newborn and expulsion of the placenta. Calcium is one of the essential materials in the body which was found that subclinical hypo-calcemia can highly affect cows and about 47% of the multiparous with 25% of the heifers were affected. Forty animals were selected and divided into two equal groups, first group treated with intramuscular administrations of calcium, the remained group were injected Intramuscular normal saline. The result showed there are significant decrease in the time of parturition in group administered with calcium as compared with non-treated group. Aim of Study: Evaluate the role of calcium on time of parturition period and some trouble shot during it.

Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage

Article

Full-text available

Sep 2020

The use of cooled semen is relatively common in goats. There are a number of advantages of cooled semen doses, including easier handling of artificial insemination (AI) doses, transport, more AI doses per ejaculate, and higher fertility rates in comparison with frozen AI doses. However, cooled semen has a short shelf life. The objective of this study was to examine the effect of temperature and sperm concentration on the in vitro sperm quality during liquid storage for 48 h, including sperm motility and kinetics, response to oxidation, mitochondrial membrane potential (MMP) and DNA fragmentation in goats. Three experiments were performed. In the first, the effects of liquid preservation of semen at different temperatures (5 °C or 17 °C), durations (0, 24 and 48 h) and sperm concentrations (250 × 106 sperm/mL (1:2 dilution rate), 166.7 × 106 sperm/mL (1:3 dilution rate) or 50 × 106 sperm/mL (1:10 dilution rate)) on sperm motility and kinetics were studied. In the second experiment, the effect of temperature, sperm washing and concentration on sperm motility and DNA fragmentation was studied. Finally, the effect of sperm concentration and duration of storage at 5 °C on sperm motility, response to oxidative stress and MMP was examined. We found that refrigerated liquid storage of goat sperm impaired sperm quality, such as motility, MMP and response to oxidation, as storage time increased; however, sperm DNA fragmentation index was not significantly affected. Liquid storage at 5 °C preserved higher total motility than at 17 °C. Moreover, we observed that the reduction of sperm concentration below 500 × 106 sperm/mL did not seem to improve the quality of spermatozoa conserved in milk-based extender in the conditions tested.

Supplementation of green tea extract (GTE) in extender improves structural and functional characteristics, total antioxidant capacity and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

Article

Full-text available

Oct 2019
THERIOGENOLOGY

SOYMILK-BASED EXTENDER FOR CRYOPRESERVATION OF BOVINE SEMEN

Article

Full-text available

Feb 2010

Red pine ( Pinus brutia Ten ) bark tree extract preserves sperm quality by reducing oxidative stress and preventing chromatin damage

Article

Full-text available

Apr 2020
ANDROLOGIA

This study aimed to investigate the effectiveness of using red pine bark tree extract (P; Pinus brutia Ten) as a TRIS extender in an attempt to prevent oxidative stress in bull spermatozoa after freezing. Semen specimens were obtained from Simmental bulls via an artificial vagina and pooled. They were separated into five specimens and diluted with Tris extender consisting of P (200, 100, 50 and 25 µg/ml) and P free (control; C) up to a final concentration of 16 × 10⁶ per straw. All specimens were equilibrated for a period of 4 hr at a temperature of 4°C, following which they were filled in 0.25‐ml French straws and frozen. Addition of P resulted in favourable tail length in comparison with C (p < .05). The lowest malondialdehyde levels and the highest glutathione levels were detected in all P groups (p < .05). Supplementation with P did not show advanced results in terms of total, progressive sperm motility and total abnormality in comparison with C (p > .05). In conclusion, it has been shown that although P added to a Tris extender does not have a positive effect on sperm motility, it prevents chromatin damage by reducing oxidative stress, in addition to reducing head abnormalities when used at the amount of 50 μg/ml.

Effect of vitrification on spermatozoa quality in bull semen

Article

Jan 2020

Cryopreservation of spermatozoa is part of the assisted reproductive biotechnology to enhance reproductive capacity in livestock. Conventional cryopreservation applies slow-gradual freezing method permitted ice crystallization and causes cryodamage resulting in poor post-thawed semen quality. Hence, vitrification is introduced by solidifying the solution into glassy state without causing any crystallization in fast and inexpensive manner. This ultra-rapid cooling method requires high concentration of cryoprotectants that potentially toxic the spermatozoa. Therefore, this study was conducted to determine the effect of vitrification on the quality of bull semen. A total of 24 bull semen samples were collected using an electro-ejaculation technique. Tris-based extender was compared with vitrification using solution of different concentration of cryoprotectants at 10% (Vitrification Solution 1; VS-1) and 20% (VS-2) containing dimethyl sulfoxide (DMSO) and ethylene glycol respectively. The result revealed that high mortality and nearly zero motility in all post-warmed vitrified spermatozoa, but the general and progressive motilities parameters in VS-1 at initial evaluation was 22.45% and 24.87% respectively better than Tris-based extender and was statistically significant. In conclusion, vitrification has potential as an alternative for cryopreservation. Therefore, further research on spermatozoa vitrification technique on enhancement in cooling and warming should be conducted and investigated. GS (2020) Effect of vitrification on spermatozoa quality in bull semen. Eurasia J Biosci 14: 3897-3904.

Directional Freezing of Cell-Seeded Electrospun Fiber Mats for Tissue Engineering Applications

Chapter

Jan 2021

As novel tissue engineered constructs (TECs) are developed, current tissue banking practices need better control over ice formation and growth to prevent cryodamage to cells and a scaffold. Directional solidification demonstrates benefits in adhered cells and native tissues cryopreservation through controlled heat transfer. Therefore, this study aims to investigate the feasibility of using this technique for cryopreservation of cell-seeded electrospun fiber mats as model TECs. Fiber mats were produced using blend electrospinning of polycaprolactone (PCL, 200 mg/ml) and poly-L-lactic acid (PLA, 100 mg/ml) dissolved in 2,2,2-Trifluoroethanol. The fiber size and morphology was characterized using scanning electron microscopy. Specific heat measurements were conducted using differential scanning calorimetry. The square-shaped fiber mats were seeded under static conditions with HeLa cells and cultivated for 24 h. Samples were directionally frozen in a sandwich format either in 10% DMSO or culture medium with the sample movement at 30 μm/s through the predetermined temperature gradients along a 2.6 mm slit. After directional solidification, samples were gradually frozen at 1 K/min down to −80 ℃. Crystal shape was visualized using cryomicroscopic system. Before freezing and 24 h after thawing, cell viability was assessed using live-dead assay. Within randomly orientated PCL-PLA fibers, HeLa cells exhibited typical shape and attachment with higher than 90% viability prior to freezing. While up to 80% of HeLa cells were alive on fiber mats after freezing using DMSO with or without directional solidification step. The demonstrated controlled freezing may assist optimizing the freezing of more sensitive cells. The results suggest that directional freezing becomes a viable option for cryopreservation in tissue engineering applications.

Nano-depletion of acrosome-damaged donkey sperm by using lectin peanut agglutinin (PNA)-magnetic nanoparticles

Article

Apr 2020
THERIOGENOLOGY

Lectin is considered as a suitable biomarker for nano-depletion of acrosome-damaged sperm. The aim of this study was to synthetize magnetic nanoparticles (MNPs) coated by peanut (Arachis hypogaea) agglutinin lectin (PNA) and investigate its beneficial effect in improving of sperm characteristics. MNPs were obtained by co-precipitation method, functionalized with chitosan and coated by PNA at a concentration of 0.04 mg/mL. Semen was frozen either with glycerol-based or sucrose-based extenders. Frozen-thawed straws from five donkeys (three ejaculates per donkey) were incubated with lectin-MNPs (2 mg/mL), and then exposed to an external magnet enabling the non-bound sperm to be collected as nanopurified sperm. Sperm were evaluated post-thawing (control) and after nanopurification for motility, plasma membrane integrity, acrosome integrity, morphology, DNA fragmentation and concentration. The statistical analyses were extended to investigate the correlation between the initial quality of the frozen-thawed semen samples and the effect of nanopurification after thawing. The obtained MNPs were biocompatible to the sperm and significantly improved the progressive motility (P < 0.05) for the glycerol nanopurified group (43.08 ± 3.52%) in comparison to control (33.70 ± 2.64%). Acrosome-damaged sperm were reduced (P < 0.05) in both nanopurified groups (19.92 ± 2.69 for G and 21.57 ± 2.77 for S) in comparison to control (36.07 ± 3.82 for G and 35.35 ± 3.88 for S). There were no significant changes in sperm morphology and membrane integrity after nanopurification. The average sperm recovery after nanopurification was 80.1%. Sperm quality index was significantly higher (P < 0.001) in nanopurified groups regardless of the initial quality of the frozen thawed semen samples. However, in the high sperm quality group, nanopurification significantly improved the progressive motility and membrane integrity besides the increasing of acrosome-intact sperm. Sperm nanopurification using lectin-magnetic nanoparticles can be considered as a suitable method to reduce the proportion of acrosome-damaged sperm and to increase the quality of frozen thawed donkey semen.

Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders

Abstract

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