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-Effect of seminal plasma on pool of cryopreserved semen from four Saanen bucks on the MTRTF, MTRTS1, VTRTS1 and MTRTS2 traits.
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The objective of this study was to evaluate the effects of egg yolk and seminal plasma on the viability of cryopreserved goat semen. To this end, four fertile Saanen bucks, aged between 10 months and 1 year, and weighing 18 to 25 kg, were used. Semen was collected from each buck by the artificial vagina method at the end of breeding season (June-Ju...
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... Pada akhir musim kawin, tingkat phospholipase A pada plasma semen akan lebih rendah dari pada saat di luar musim kawin. Selain itu umur ternak juga mempengaruhi kandungan phospholipase A. pada ternak muda, yang sistem reproduksinya masih berkembang (umur 10 bulan sampai 1 tahun) memiliki produksi phospholipase A yang lebih rendah daripada ternak dewasa, yang kelenjar bulbourethralisnya telah berkembang (Ferreira et al., 2014). ...
Penggunaan pengencer komersial pada pembuatan semen beku kambing PE umum digunakan di Indonesia. Namun penggunaan pengencer komersial ini memiliki kelemahan, yaitu masa simpan yang pendek dan keterbatasan akses perolehan barang yang tidak tersedia setiap saat, terutama untuk balai inseminasi buatan daerah (BIBD). Untuk itu perlu diketahui tentang pengaruh beberapa jenis pengencer berbasis skim dan tris yang telah dikembangkan terhadap kualitas semen cair dan semen beku kambing PE, sehingga dapat menjadi alternatif ketika pengencer komersial tidak tersedia. Penelitian ini menggunakan Rancangan Acak Kelompok (RAK) dengan perlakuan 5 jenis pengencer yaitu : P1 : Pengencer Andromed (Andromed) , P2 : Pengencer Skim + Kuning Telur (SKT), P3 : Pengencer Skim + 1% Soybean lecithin (SSL), P4 : Pengencer Tris + Kuning telur (TKT), P5 : Pengencer Tris + 1% Soybean lecithin (TSL). Semen segar yang digunakan berasal dari 1 ekor kambing PE yang dipelihara dan dikoleksi semennya sesuai SOP BIB Ungaran. Hasil analisis statistik menunjukkan bahwa jenis pengencer berpengaruh nyata terhadap motilitas, viabilitas dan persentase membran plasma utuh semen cair dan semen beku kambing PE (P <0,05). Motilitas, viabilitas dan persentase membran plasma utuh tertinggi didapatkan pada perlakuan jenis pengencer Andromed (P1) dan yang terendah pada perlakuan pengencer Skim-Soybean Lecithin (P3). Namun pada kondisi pengencer komersial tidak tersedia, jenis pengencer berbasis skim dan tris dengan kuning telur dapat dipergunakan sebagai alternatif pengencer untuk pembuatan semen beku kambing PE.
... However, washing sperm removes the antioxidative enzymes and protective enzymes, whereas the egg yolk concentration influences the cryoprotective ability of the semen extender [18]. So it indicates that the various extrinsic and intrinsic factors regulate the standard of sperm during different stages of cryopreservation. ...
... The various concentrations of tris and egg yolk were added separately to the frozen semen extenders of different animals in the different studies and affected semen quality after freezing and thawing [18][19][20]. However, the synergistic effects of using different combined concentrations of tris and egg yolk supplementation in the freezing extender for buck spermatozoa have not been well explained. ...
Objectives: This study was designed to examine the effects of various concentrations of tris (hydroxymethyl) aminomethane (tris) and egg yolk on the quality of cryopreserved buck sperm.
Materials and Methods: The collected semen samples were pooled, washed, and diluted into five different freezing extender groups, viz., extender I (tris 0% + egg yolk 0%), extender II (tris 1.41% + egg yolk 4%), extender III (tris 2.41% + egg yolk 8%), extender IV (tris 3.41% + egg yolk 16%), and extender V (tris 4.41% + egg yolk 24%). The sperm parameter of the five groups of extenders was evaluated after equilibration and cryopreservation.
Results: The results showed that extenders II–V provided significantly higher semen progressive motility and total motility percentages than extender I after equilibration (p < 0.05). The higher percentages of semen progressive motility, total motility, viability, and plasma membrane integ¬rity (by both HOST under light microscopy and stain after HOST under light microscopy) were found in the sperm cryopreserved with extender IV than extender I, extender II, and extender III groups after thawing (p < 0.05). In addition, semen progressive motility, total motility, and viability were not further increased, or plasma membrane integrity (by both HOST tests) was decreased by the addition of tris and egg yolk (extender V) after cryopreservation (p < 0.05).
Conclusion: In conclusion, our result indicates that the following washing, the supplementation of tris (3.41% + egg yolk 16%) on the freezing extender are suitable for improving the semen quality of buck after freezing and thawing. [J Adv Vet Anim Res 2022; 9(4.000): 676-683]
... Hence, removal of the seminal plasma is important to improve the quality of frozen-thawed buck semen. Some of the centrifugation regimes and washing solutions evaluated earlier for goat semen cryopreservation were 800 g for 15 minutes with Tris citric acid buffer (Tuli and Holtz, 1994;Ferreira et al., 2014), 600 g for 10 minutes with Krebs-Ringer phosphate plus sodium citrate (Azeredo et al., 2001), 1200 g for 15 minutes with Tris citric acid glucose (TCG) buffer (Peterson et al., 2007), 1500 g for 10 minutes with TCG (Kozdrowski et al., 2007) and 1000 g for 10 minutes with Ringer's lactate (Sariozkan et al., 2010). However, there was no unanimity regarding the best centrifugation regime for yielding superior quality semen. ...
... Sen (2015) recorded 51.01.56 per cent post-thaw sperm motility in Norduz goat semen frozen using 10 per cent egg yolk in skimmed milk following centrifugation at 600 g for 10 minutes. Ferreira et al. (2014) found 13.47% post-freeze sperm motility on cryopreservation of buck semen centrifuged at a speed of 800 g for 15 minutes and diluted in Tris citrate extender with 10 per cent egg yolk. When the mean live sperm was compared, that obtained in the experiment performed by Cabrera et al. (2005) after freezing Canary buck semen by centrifugation at 700 g for 15 minutes was 17.9 per cent which was much lower than the present findings. ...
Background: The present study documents the association of repeat breeding and silent estrus in cows with iodine deficiency, hypothyroidism and abnormal metestrus bleeding in high-producing Jersey and Holstein Friesian crossbred cows of Assam in India. Methods: The field based study was conducted involving 470 Holstein Friesian and Jersey crossbred cows in Assam of North East India during the period August, 2021 to July, 2022. Out of 470 crossbred cows investigated, 62 (13.00%) with repeat breeding and abnormal metestrus bleeding (Group-A), 18 (3.83%) were diagnosed as silent estrus (group-B) and 6 with normal breeding history (Group-C) without the signs of metestrus bleeding were randomly selected for the study. The cows belonging to all the 3 groups were subjected to serum T3, T4 and iodine estimation. Result: 42 (67.74%) crossbred cows in group A (n=62) and 13 (72.22%) in group B (n=18) were diagnosed to have hypothyroidism including Iodine deficiency. The overall incidence of hypothyroidism out of 470 cows was 11.70%. Group C showed significantly higher T3 and T4 levels than groups A and B. Thus, it concluded that iodine deficiency -induced hypothyroid condition might be attributed to the causes of abnormal metestrus bleeding associated with repeat breeding and silent estrus in high-producing crossbred cows of Assam in India.
... Hence, removal of the seminal plasma is important to improve the quality of frozen-thawed buck semen. Some of the centrifugation regimes and washing solutions evaluated earlier for goat semen cryopreservation were 800 g for 15 minutes with Tris citric acid buffer (Tuli and Holtz, 1994;Ferreira et al., 2014), 600 g for 10 minutes with Krebs-Ringer phosphate plus sodium citrate (Azeredo et al., 2001), 1200 g for 15 minutes with Tris citric acid glucose (TCG) buffer (Peterson et al., 2007), 1500 g for 10 minutes with TCG (Kozdrowski et al., 2007) and 1000 g for 10 minutes with Ringer's lactate (Sariozkan et al., 2010). However, there was no unanimity regarding the best centrifugation regime for yielding superior quality semen. ...
... Sen (2015) recorded 51.01.56 per cent post-thaw sperm motility in Norduz goat semen frozen using 10 per cent egg yolk in skimmed milk following centrifugation at 600 g for 10 minutes. Ferreira et al. (2014) found 13.47% post-freeze sperm motility on cryopreservation of buck semen centrifuged at a speed of 800 g for 15 minutes and diluted in Tris citrate extender with 10 per cent egg yolk. When the mean live sperm was compared, that obtained in the experiment performed by Cabrera et al. (2005) after freezing Canary buck semen by centrifugation at 700 g for 15 minutes was 17.9 per cent which was much lower than the present findings. ...
Background: Goat semen has phospholipase A enzyme which makes removal of seminal plasma an important step in its preservation. This study was aimed to standardize a centrifugation regime for cryopreservation of Beetal buck semen. Methods: A total of 36 pooled semen ejaculates were used for the experiment where initially 27 pooled ejaculates were utilized to find out the best time period out of 5, 8 and 11 minutes for each g-force viz., 700 x g, 1100 x g and 1400 x g using 9 pooled ejaculates per g-force. The remaining 9 pooled ejaculates were employed to find out the best centrifugation regime amongst the best time period for each g-force (8 minutes for 700 x g, 8 minutes for 1100 x g and 5 minutes for 1400 x g). Result: The percentage of sperm motility, live sperm, intact acrosome and HOST-reacted sperm differed significantly (P less than 0.0001) between time periods at 700 x g, 1100 x g and 1400 x g. The sperm parameters were significantly (P less than 0.05) higher at 1400 x g for 5 minutes and 1100 x g for 8 minutes than at 700 x g for 8 minutes. In conclusion, adoption of a high centrifugation force for a short duration of time for washing could improve the quality of frozen spermatozoa.
... However, other researchers reported better results with noncentrifuged goat semen with an extender containing egg yolk when compared with centrifuged semen. 7,8 Elimination of buck seminal plasma by centrifugation of semen was useful for the survival of spermatozoa after freeze-thawing. The process reduced the deleterious effects of phospholipase A in egg yolk containing diluent. ...
Sperm motility, normal morphology, viability, spermatozoa DNA damage, and lipid peroxidation are all affected by semen cryopreservation. The goal of this study was to see how effective cupric oxide nanoparticles (CuONPs) are as a cryo-extender additive on post-thawed sperm parameters. An artificial vagina was used to collect semen samples from five mature Zaraibi bucks (2-3 years). Ejaculates were pooled and separated into two fractions (A&B), a fraction (A) was left without being centrifuged and a fraction (B) was centrifuged to remove seminal plasma. Both fractions were diluted with tris egg yolk citrate extender (TECE) and then divided into five equal aliquots, each supplemented with (0, 10, 20, 40, and 60 ppm/ml) CuONPs. The findings revealed that removing seminal plasma before cryopreservation harms sperm parameters. Sperm motility, viability index, membrane integrity, biochemical antioxidant marker, DNA integrity, and MDA level improved after supplementation with CuONPs up to 60 ppm/ml, the most prominent significant positive effect was obtained with the highest dose (60 ppm/ml) without removal of the seminal plasm compared to control group. In conclusion: The presence of seminal plasma with a high concentration of CuONPs (up to 60 ppm/ml) may help to mitigate the negative effects of cryo-preservation.
... Therefore, the removal of seminal plasma by washing (centrifugation) in an isotonic medium is beneficial for the survival of spermatozoa during cryopreservation. 17,18 However, during washing, along with detrimental phospholipase A (EYCE), the beneficial elements of seminal plasma are also removed. Seminal plasma has a key role in sperm function, fertilization, and embryonic development in the female reproductive tract. ...
The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the extender before cryopreservation. In a preliminary experiment, different levels of BSP were supplemented (1, 3, 5, 7, and 9% v/v) in egg yolk (7.5% egg yolk)-tris (EYT) extender and used for cryopreservation of Pantja buck semen. Results in terms of motility, viability, plasma membrane integrity, acrosome integrity, and lipid peroxidation showed that 5% BSP was suitable for maintaining Pantja buck semen quality during cryopreservation. In the final experiment, pooled semen from four Pantja bucks was split into three aliquots (I, II, and III). Aliquot I was directly diluted in EYT extender and grouped as the control (C); aliquot II and III were washed separately with TALP solution and diluted as D1 (Washed semen with EYT extender) and D2 (Washed semen with EYT extender containing 5% BSP), respectively. Seminal attributes (sperm individual motility, viability, plasma membrane integrity, acrosome integrity, and total morphological abnormalities) were assessed at the postdilution, postequilibration, and post-thawing stages. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) concentration, and glutathione peroxidase (GSH-Px) activity were measured at post-thaw. Washed semen significantly improved (p < 0.05) seminal parameters at post-thaw compared with unwashed semen (control). A significant difference (p < 0.05) was observed in seminal attributes between freezing stages and between dilution groups. Significantly higher (p < 0.05) post-thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, and GSH-Px activity, and significantly lower (p < 0.05) MDA concentration and extracellular release of enzymes (ALT, AST) were observed in group D2 compared with control and D1. The results of the present study demonstrated that cryopreservation of washed Pantja buck semen diluted with 5% BSP-supplemented EYT extender can improve post-thaw semen quality.
... In relation to goat semen, the negative interaction between seminal plasma rich in phospholipase A2 and egg yolk and/or skim milknonpenetrating cryoprotectants traditionally used in the semen extenderis widely known (Forouzanfar et al., 2010;Purdy, 2006), a fact that leads to the need for removal of seminal plasma prior to cryopreservation (Machado and Simplício, 1995). When eliminating the seminal plasma, the antioxidant systems present in it are also removed and the goat sperm becomes even more susceptible to ROS during the cryopreservation process (Bilodeau et al., 2000;Ferreira et al., 2014); thus, melatonin is presented to be a potential alternative due to its antioxidant capacity, when there is a seminal plasma elimination. ...
The objective of this study was to investigate the effect of melatonin supplement in different extenders on the quality of cryopreserved goat semen. Six semen pools were cryopreserved in skim milk (SM - Experiment 1) or tris-egg yolk (TEY- Experiment 2) based extenders supplemented with melatonin (0, 0.5, 1, 2 and 4 mM). After thawing, the kinetic sperm parameters [total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), straightness (STR), wobble (WOB)] and viability parameters [plasma and acrosomal membrane integrity (iMPA), high mitochondrial membrane potential (hPMM) and intracellular ROS production (iROS)] were observed at time 0 hours and after 2 hours of incubation (37 °C). In the experiment I, only for the hMMP variable (p <0.05), a melatonin vs. time interaction was observed. Was noted a negatively influenced in the variables over the incubation period, except for the STR, iMPA, hMMP and iROS parameters. The treatment with melatonin had different effects in the variables VCL, VSL, WOB, hPMM, and iROS, however, in the variables TM, PM, LIN, STR and iMPA there was no significant difference regarding the different melatonin concentrations. In the experiment II, no interaction was observed, melatonin vs. time, for the analyzed variables. Was noted a negatively influenced (p <0.05) in all variables throughout the incubation period, with the exception of the STR, iMPA and hMMP parameters. The treatment with melatonin showed different effects in the variables TM, PM, VSL, LIN, STR, WOB, iMPA, hPMM and iROS, however, in the variable VCL there was no significant difference regarding the different concentrations of melatonin. In conclusion, melatonin does not improve the post-thawing quality of goat semen cryopreserved in SM or TEY based extenders and may be deleterious when supplemented at concentration of 4 mM.
... Ejaculation volume was assessed directly from a graduated collection tube, and the pH of the semen samples was measured using pH-indicator strips immediately after the semen collection. Progressive sperm motility was evaluated by depositing a drop of semen diluted (1:10) in pre-warmed egg yolk citrate on a warm cover slide and cover slip 66 . Motility was evaluated as the proportion of forward-moving sperm cells under an optical microscope with a 40 × magnification by two experienced veterinarians. ...
This study determined the effects of scrotal insulation on testicular functions in bucks and evaluated the impact of exogenous gonadotropin-releasing hormone (GnRH) administration before scrotal insulation on sperm production and testicular vascular dynamics. Twelve bucks were randomly divided into three groups: scrotal-insulated animals without GnRH treatment (INS), scrotal-insulated animals treated previously with GnRH (GnRH + INS), and animals without insulation as controls (CON). Doppler ultrasonography was used to evaluate testicular vascular changes, and semen samples were collected to assess seminal parameters. Testicular samples were collected from slaughtered bucks at the end of the experiment for histological investigations and immunohistochemical analysis for caspase 3 (apoptotic marker), and a vascular endothelial growth factor (VEGF; hypoxic marker) evaluation. Sperm motility drastically decreased (33%) in the INS group on day 8 compared with those in the GnRH + INS and CON groups (58% and 85%, respectively). Testicular blood flow significantly decreased for 3 and 2 weeks in the INS and GnRH + INS groups, respectively. The pulsatility index (PI) reached pretreatment values at 5 and 4 weeks after insulation in the INS and GnRH + INS groups, respectively. The resistance index (RI) values increased in both insulated groups for the first 2 weeks and decreased to control values 4 weeks after insulation. However, the maximum velocity (VP) started to increase reaching pretreatment values by the 5th and 3rd weeks after insulation in the INS and GnRH + INS groups, respectively. Histological investigations showed a marked reduction in lipid inclusions in Sertoli cells in the GnRH + INS group compared with those in the INS group. The distributions of both caspase 3 and VEGF decreased in the GnRH + INS group compared with those in the INS group. This study showed that the administration of a single dose of GnRH delayed the negative effects of scrotal insulation on different seminal traits and revealed the pivotal role of GnRH in compensating testicular insulation in bucks.
... Este comportamiento podría estar influenciado por la fosfolipasa A presente en el plasma seminal, que hidroliza la lecitina de la yema de huevo y produce ácidos grasos y lisolecitina, los primeros, causan la disminución del pH del semen diluido, lo cual reduce la viabilidad de los espermatozoides, mientras que la lisolecitina tiene un efecto tóxico sobre la motilidad de los espermatozoides y la integridad de la membrana celular espermática, lo que también reduce la capacidad para fertilizar, esta reacción enzimática es más intensa en la medida que aumenta la concentración de YH (Dhaher y Aziz, 2021; Moreno et al., 2021;Sharma y Sood, 2020). Resultados similares fueron observados por Ferreira et al. (2014), quienes señalan que un 5% de YH proporcionó mejores resultados de motilidad y viabilidad, en comparación con el semen procesado en un diluyente con un 10% de YH sin realizar lavado seminal. ...
pre> Background: Prior to freezing goat semen, it is necessary to perform seminal washing by centrifugation to eliminate Phospholipase A, with the consequent loss of elements involved in maintaining sperm functions. Objective: Determine the adequate concentration of egg yolk (YH) for freezing goat semen in a lyophilized diluent based on Tris-glucose and citric acid, without performing seminal washing by centrifugation. Methodology: ninety ejaculates were evaluated with 12 replicates, collected twice a week by means of Artificial Vagina. Volume, motility, concentration, viability and total pathologies were measured. The fit ejaculates were united and divided into five portions, each one received the corresponding diluent: Tris-glucose-Ac. Citrus with YH (2.25%, 3.37%, 4.45% and 5.65%) and the control diluent containing lactose-skimmed milk (DC). The final sperm concentration in the samples was 1.5 x 109 mL <sup>-1</sup>. The equilibrium period was carried out at 5°C for 2 h. Subsequently, it was frozen in 0.1 mL tablets in nitrogen vapors, and stored for 7 d in liquid nitrogen, thawed at 37°C and the percentages of motility (30 min, 120 min and 240 min), viability and total pathologies (30 min and 120 min) were determined. The diluents were compared using a Binary Logistic Regression model. Results: YH (4.45%) and DC had the highest probability (P <0.05) of motility at all times. The highest probability (P <0.05) of viability was for YH (4.45%), and the lowest probability (P <0.05) of total pathologies for 4.45% YH and DC, at 30 min and 120 min. Implications: In the freezing of goat semen, it is possible to eliminate the seminal washing process by centrifugation. Conclusion: Goat semen can be frozen in a Tris-based lyophilized extender with 4.45% egg yolk, without performing seminal washing by centrifugation.
... The high percentage of total acrosome damage (dead and viable sperm) seen in sperm incubated in IVF media in the presence of SP cannot be explained due to the incidence of spontaneous acrosome reaction, as discussed earlier. In this regard, a potential explanation may be that buck seminal plasma could cause acrosome damage due to the presence of a hydrolysing enzyme, phospholipase-A2, found in buck seminal plasma [28,29]. Nevertheless, our results are in agreement with a report in ram [30] demonstrating that the addition of SP did not improve the sperm plasma membrane integrity and acrosome status, despite the fact that seminal plasma has been reported to reverse cold shock [31,32]. ...
In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.
