Frozen Semen Research Articles

Effects of k-carrageenan supplementation or in combination with cholesterol-loaded cyclodextrin following freezing-thawing process of rooster spermatozoa

This experimental research purposely seeks to explore the effect of supplementing k-carrageenan (k-CRG) or CLC (cholesterol-loaded cyclodextrins) or the combined effect of k-CRG and CLC as supplements of antioxidants to an extender for rooster semen freezing. A total of 75 neat pooled ejaculates were collected twice a week from twenty-five (25) commercial line arbor acres broiler roosters (30 wks) during the experimental period. In each replicate, semen samples (n= 15, three ejaculates per rooster) were pooled and divided into nine equal aliquots, and each aliquot was diluted with one of the following extender supplemented with k-CRG, CLC, and k-CRG + CLC after which it was subjected to cryopreservation process using the “pellet” method. In study I, the supplementation of extenders with k-CRG was in five equal aliquots as follows; (0.2, 0.4, 0.6, 0.8) mg/mL and control group (k-CRG 0) mg/mL while in Study II, there was a combination of both k-CRG + CLC (0.4 mg/mL + 1.5 mg/mL, respectively), 0.4 mg/mL k-CRG, 1.5 mg/mL CLC and control group. Sperm quality parameters, endogenous antioxidant enzymes, lipid peroxidation (MDA) and ROS were all assessed after the freeze-thaw process. Our findings in study I indicated that at post-thaw, an optimum 0.4 mg/mL k-CRG supplementation in the extender improved semen quality parameters, endogenous enzymes, MDA and ROS in comparison to the control group. Interestingly prior to the freeze-thaw process, it was depicted in study II that combined k-CRG + CLC (0.4 mg/mL+1.5 mg/mL) inclusion in the extender provided maximum protection to sperm quality parameters, endogenous enzymes, MDA and ROS in comparison to 1.5 mg/mL CLC and control group at post-thaw. Besides, there was also a significant difference observed in the extenders supplemented with combined k-CRG + CLC (0.4 mg/mL +1.5 mg/mL) when compared to 0.4 mg/mL k-CRG for semen quality parameters and endogenous antioxidant enzymes (SOD, CAT, and GPx) but no significant difference was observed for MDA and ROS. Also, there was a significant difference observed in the extender supplemented with 1.5 mg/mL CLC when compared to the control group for semen quality parameters, SOD, CAT, and MDA but no significant difference for GPx and ROS at post-thaw. In conclusion, k-CRG at an optimal dosage of 0.4 mg/mL proved effective for improving post-thaw sperm quality but its combined addition k-CRG + CLC at an optimal concentration of (0.4 + 1.5) mg/mL in the extender provided greater protection to the rooster spermatozoa at post-thaw.

First Evaluations and Cryopreservation of Semen Samples from Sunda Clouded Leopards (Neofelis diardi).

Simple SummaryThe Sunda clouded leopard is a medium-sized cat from Borneo and Sumatra. The captive population has less than 20 individuals on both islands. Conservation breeding of this species has been unsuccessful due to the small numbers of individuals and the difficulty of pairing them. The present study aimed at evaluating for the first time the reproductive organs, semen characteristics, and sperm freezing for two wild-caught males (8 and 11 year old) living singly. The morphometric and ultrasound examination showed some degree of abnormalities in both leopards. Semen evaluation obtained by electro-ejaculation revealed a high proportion of abnormal sperm cells (similar to mainland clouded leopards). While only the younger male produced samples that could be frozen, post-thaw evaluations showed that the few surviving spermatozoa could potentially be used for in vitro fertilization or sperm injection. Studies on more individuals are needed to validate those encouraging but preliminary results.A better understanding of semen characteristics and resilience to freezing temperatures is necessary before developing assisted reproductive techniques and systematic biobanking for the Sunda clouded leopard. The objective of this study was to evaluate for the first time the semen and sperm quality (in fresh and frozen samples) of two captive Sunda clouded leopards in Malaysia. A total of 17 examinations of the reproductive tract (using ultrasonography) and electro-ejaculations were performed on the two leopards over a 2-year period. Samples obtained from Leopard 1 (8 years old) varied in terms of volume (402 ± 92 µL), pH (7.9 ± 0.9), sperm motility (54.5 ± 24.2%), sperm concentration (122.4 ± 84.7 × 106 sperm/mL), normal morphology (23.9 ± 12.3%), and viability (55.2 ± 18.2%). Midpiece defects represented the most common structural abnormality followed by abnormal tail and head defects. Samples from Leopard 2 (11 year old with abnormal testicular tissue) were of lesser quality. Two frozen semen samples from Leopard 1 were thawed and examined for acrosome integrity. Post-thawed samples contained <10% of motile spermatozoa but almost 50% of abnormal acrosomes. The present results emphasized the high incidence of structurally-abnormal spermatozoa, similar to the mainland clouded leopard. Post-thaw evaluations showed that the few surviving spermatozoa could potentially be used for in vitro fertilization or sperm injection. However, more individuals must be studied to validate those first findings that are exciting but still preliminary.

Post-thawing Sperm Quality in Chilean Purebred Stallions: Effect of Age and Seasonality

In this work, we investigated the influence of age and seasonality on sperm motility and DNA fragmentation in post-thawing semen from Chilean Purebred Stallions (CPS), a horse breed presenting the oldest genealogy record in South America with an interesting reproductive industry. Despite that semen from aged CPS is frozen all year round, there is a lack of studies characterizing the breed semen freezability in accordance with age and seasonality. Twenty fertile CPS were grouped into the young group, the middle group, and the aged group. Ten ejaculates from each stallion were obtained by using an artificial vagina during summer (December) and winter (July) and directly frozen. Subsequently, the frozen semen was thawed and analyzed by a computer-assisted semen analysis and flow cytometer assessing progressive motility, mean velocity, and DNA fragmentation spermatozoa. Kruskal–Wallis test and Pearson’s correlation were used to determine statistical differences among groups and correlation among variables (P ≤ .05). Both spermatozoa motility traits decreased progressively in accordance with age and seasonality, showing the lowest values in the aged group during winter and the highest values in the young group during summer. Deoxyribonucleic acid fragmentation increased significantly in accordance with age and seasonality being highest in the aged group during winter and lowest in the young group during summer. Post-thawing sperm quality showed a negative correlation with the age of the stallions and a positive correlation with the normal sperm morphology before freezing. These results allow the conclusion that age and seasonality are important factors that need to be considered during the selection of CPS for reproductive programs.

Effect of relaxin on cryopreserved beef bull semen characteristics

This study aimed to improve the quality of cryopreserved beef bull (Piedmontese) semen by incorporation of relaxin in diluted semen before cryopreservation procedures. Semen samples were collected from 4 proven fertile bulls, using artificial vagina, once per week for 8 consecutive weeks and pooled together then diluted with Bullxcell® extender, and supplemented with different concentrations of relaxin (0 (control), 25, 50 and 100 ng/ml) before cooling, equilibration and freezing procedures. Frozen semen was thawed and assessed for motility by Computer-Assisted Sperm Analysis and vitality parameters such as acrosome, plasma membrane and DNA integrities, apoptosis, mitochondrial membrane potential, mucus penetration and SOD activity. The developmental potential of bovine embryos produced in vitro by using relaxin-treated was also investigated. In the present study, 50 and 100 ng/ml relaxin incorporation in extended bull semen before cryopreservation induced a reduction of sperm motility immediately after thawing (0h), whereas, during long incubation periods (1–2 h), relaxin showed a significant positive effect on sperm quality by improving the sperm motility and velocity parameters. Interestingly, sperm vitality was improved by 25 and 100 ng/ml relaxin and the blastocyst developmental rate was significantly increased in the 25 ng/ml relaxin group compared with controls (52/118, 44.0% vs. 32/116, 27.6%, respectively). These findings suggest a potential use of relaxin at the doses tested in the present study as an additive in the cryopreservation media of bull semen to improve sperm quality.

Resveratrol supplementation and cryopreservation of buck semen

Resveratrol (Resv) was tested to assess its effects on buck semen freezability. Ejaculates of 4 bucks were collected, washed and diluted in a commercial extender at 30 °C. Extended semen was divided into 4 aliquots supplemented with increasing concentrations of Resv: 0 μM (control); 10 μM; 25 μM and 50 μM. Aliquots were cooled to 4 °C in 5h and frozen in LN2. Thawing was performed at 37 °C for 30 s. At the 3 stages of the experiment (30 °C, 4 °C, thawing), motility (CASA), osmotic resistance (Hos test) and integrity of cytoplasm and acrosome membranes (PI/PSA staining) were assessed. Moreover, in thawed samples, the oxidative status (MDA assay) and early apoptosis (DNA fragmentation by TUNEL assay) were evaluated. Resveratrol supplementation did not affect most of the motility parameters analysed, except for total motility, ALH (lateral head displacement) and velocity distribution (P < 0.05). Functional and morphological integrity of membranes was not affected at any stage of the experiment (P > 0.05). In thawed spermatozoa, the oxidative status was not preserved by Resv (P > 0.05) while early apoptosis, was significantly decreased in the 50 μM Resv group (P < 0.05). Resveratrol did not improve buck semen freezability; the observed effects on motility and DNA were not dose dependent and not mediated by a potential anti-oxidant activity.

Supplementation of cryodiluent media with seaweed or Nile tilapia skin sulfated polysaccharides for freezing of Colossoma macropomum (Characiformes: Serrasalmidae) semen

Tambaqui, Colossoma macropomum, is an ideal fish for cultivation in captivity and it is valued in the consumer market. Studies on various aspects of aquaculture, including semen freezing, have been conducted. Despite its many benefits, cryopreservation damages the samples. Thus, to refine the existing protocols, products with antioxidant properties can be added, such as sulfated polysaccharides. This study evaluated the effect of adding sulfated polysaccharides from seaweed (green: Caulerpa cupressoides; red: Acanthophora muscoides and Solieria filiformis) and Nile tilapia skin into the tambaqui semen freezing medium. The resulting polysaccharidic samples were diluted and added to the cryopreservation medium. The semen was then collected, pooled, and subsequently frozen. The pools were diluted and frozen in solutions containing powdered coconut water (ACP-104) and 10% dimethyl sulfoxide (DMSO), and were supplemented with different polysaccharide concentrations (100, 250, 500, or 1000 μg/mL) from seaweed or Nile tilapia skin. Semen cryopreserved in an un-supplemented solution was used as control. After 45 days, the samples were thawed by immersion in a water bath (45 °C/8 s) and the membrane integrity, the morphology and, the sperm motility kinetics were evaluated. There were no differences in sperm kinetics for any of the analyzed parameters that could be linked to the polysaccharide source that was used. For membrane integrity, semen supplemented with sulfated polysaccharides from Nile tilapia skin showed better results (76.3 ± 1.9%) compared to treatments supplemented with A. muscoides (65.0 ± 1.8%), C. cupressoides (66.1 ± 1.7%), or S. filiformis (68.8 ± 1.0%), but there was no difference compared to the control (73.6 ± 2.9%). For morphology, the best results were observed in semen supplemented with C. cupressoides (86.7 ± 0.8%) and S. filiformis (87.7 ± 1.0%) polysaccharides at 100 μg/mL. For the mean path velocity (VAP), semen treated with 250 μg/mL of polysaccharides was significantly faster for A. muscoides (21.1 ± 2.4 μm/s) compared to S. filiformis (15.8 ± 1.7 μm/s). However, at 1000 μg/mL, these two red algae shown to be more efficient than the green algae. In C. cupressoides, 1000 μg/mL of polysaccharides showed lower velocity (13.8 ± 1.5 μm/s) than the other concentrations. In S. filiformis, 100 μg/mL and 1000 μg/mL of polysaccharides did not differ and presented higher VAP than 250 μg/mL and 500 μg/mL. Therefore, the addition of sulfated polysaccharides extracted from aquatic sources to ACP-104 and 10% DMSO solution is safe and has a potential of applicability on C. macropomum semen cryopreservation.

HCG is more effective than the GnRH agonist buserelin for induction of the first ovulations of the breeding season in sulpiride-primed recipient mares

In this work, we investigated the influence of age and seasonality on sperm motility and DNA fragmentation in post-thawing semen from Chilean Purebred Stallions (CPS), a horse breed presenting the oldest genealogy record in South America with an interesting reproductive industry. Despite that semen from aged CPS is frozen all year round, there is a lack of studies characterizing the breed semen freezability in accordance with age and seasonality. Twenty fertile CPS were grouped into the young group, the middle group, and the aged group. Ten ejaculates from each stallion were obtained by using an artificial vagina during summer (December) and winter (July) and directly frozen. Subsequently, the frozen semen was thawed and analyzed by a computer-assisted semen analysis and flow cytometer assessing progressive motility, mean velocity, and DNA fragmentation spermatozoa. Kruskal–Wallis test and Pearson’s correlation were used to determine statistical differences among groups and correlation among variables (P ≤ .05). Both spermatozoa motility traits decreased progressively in accordance with age and seasonality, showing the lowest values in the aged group during winter and the highest values in the young group during summer. Deoxyribonucleic acid fragmentation increased significantly in accordance with age and seasonality being highest in the aged group during winter and lowest in the young group during summer. Post-thawing sperm quality showed a negative correlation with the age of the stallions and a positive correlation with the normal sperm morphology before freezing. These results allow the conclusion that age and seasonality are important factors that need to be considered during the selection of CPS for reproductive programs.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

Obtaining Frozen Semen of Bulls with Superior Qualitative Parameters

Sperm is a zootechnical production that is harvested, controlled, processed and used, within a well-defined technological stream.Biotechnological intensification of breeding to increase birth and prolificity in zootechnics requires complex control of spermatic production on scientific bases in accordance with current legislation and compliance with national and international standards.In this regard, complex control of sperm production, regardless of its origin, is required by performing ordinary and special spermograms. In the modern technological flow of artificial cattle insemination, processing by dilution is an essential step with obvious zooeconomic implications, because it greatly increases the number of usable doses obtained from an ejaculate, increases the possibility of insemination a much bigger number by larger females, directly contributing to increased male selection pressure.

Human sperm cryopreservation prior cancer treatment: Follow up of 480 males for 23 years.

e24061 Background: Cancer treatment represents a potential risk for spermatogenesis and may compromise male fertility. Semen freezing has become a routine process for male patients prior to cancer treatment to preserve their fertility. We sought to provide information regarding what happens to the cryopreserved material and the fertility in cancer treated patients. Using a cohort from a 23-year cryopreservation program. Methods: Patients whom cryopreserved sperm to preserve fertility due to diagnosis of cancer between 1995 and 2018 in a single private center of reproductive medicine in south of Brazil were selected. A retrospective analysis of medical records was performed and clinico-pathological characteristics were collected using standard templates. Results: A total of 480 patients were evaluated for sperm cryopreservation before cancer treatment. Testis cancer was the most prevalent (59.3%), followed by hematologic malignancies (21.4%) and prostate cancer (5.4%). The age at cryopreservation varied from 14 to 72 y/o. Among them, 61.3% were up to 30 years old, and 10.8% more than 40 y/o. 8 patients decided to not cryopreserve The mean time between the freezing and the beginning of cancer treatment was 5.5 days. Eleven patients presented azoospermia; 3 of them froze testicular tissue. A shorter time between cancer diagnosis and sperm banking was observed for hematologic malignancies ( p=0.044). Out of the 472 patients, 46.8% still keep the sperm cryopreserved, 34.3% decided to discard it, 1.9% transferred it to another clinic, 8,5% abandon the specimen and 8,5% died without using it. The main reason for discard the sample was pregnancy and/or normal sperm count after treatment. The mean age of discarding was 35.7±10.3 years old. The period of maintenance cryopreservation was 5 years (min 1 month, max 23 years). The mean time of freezing before abandoning was 7.5±4.4 years Eighty-two patients achieved pregnancy. Spontaneous pregnancies occurred in 53 cases, and 47 underwent assisted reproduction with the cryopreserved sample, with 61,7% of pregnancy (n=29).15.3% of those that have children still keep the sperm cryopreserved; 11.4% gave up on having children. Out of the 133, 66.9% authorized the use of their specimen post-mortem. Conclusions: Only one third of the patients have maintained fertility after cancer treatment and 35% of pregnancies after cancer treatment were possible due to sperm preservation. Considering the high pregnancy rate with assisted reproduction techniques in patients that lost testicular functionsemen cryopreservation is warranted.

Acrosome integrity and mitochondrial membrane potential of frozen thawed buffalo semen treated with heparin binding protein

The experiment was conducted to study the sperm acrosome integrity and mitochondrial membrane potential (MMP) of frozen thawed buffalo semen treated with heparin binding protein (HBP). Buffalo semen straws from 10 bulls were procured from Central Frozen Semen Production and Training Institute, Hesseraghatta, Banglore-560088. The frozen straws were thawed at 37 ºC for 30 seconds and emptied into a 15mL sterile plastic centrifuge tube containing 1mL capacitation medium (control), addition of 25µg/mL (treatment I), 50µg/mL (treatment II) and 100µg/mL (treatment III) of HBP. The contents were incubated at 37 ºC for 2 hours. After incubation, sperm acrosomal integrity was assessed by Giemsa stain method. In control, HBP treatment I, II and III, 51.50% ± 1.29, 46.30% ±0.86, 43.40% ± 0.66and 42.15% ± 0.40 spermatozoa, respectively had intact acrosome. But, 48.50% ±1.29, 53.70% ± 0.86, 56.60% ± 0.66and 57.85% ± 0.40 spermatozoa in control, HBP treatment I, II and III respectively had lost acrosome. Significantly (P

Cryopreservation of boar semen

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

Frozen semen quality and fertility of imported pure Holstein Friesian and Sahiwal breeding bulls in Bangladesh

The present study was conducted to evaluate the frozen semen quality in terms of different motility patterns and fertility of imported pure Holstein Friesian and Sahiwal breeding bulls maintained under farming condition of Bangladesh. 100% pure Holstein Friesian and Sahiwal breeding bulls were imported and thereafter reared in the American dairy limited (ADL). Semen samples were collected once a week, processed and frozen in the laboratory of ADL. On the day of collection, semen volume and sperm concentration measured immediately after collection. Different motility patterns of spermatozoa (progressive, fast, slow, local, immotile) were measured by Computer Assisted Semen Analyzer (CASA). Effect of breed was found significant (p&lt;0.05) on semen volume and sperm concentration. The semen volume (ml/ejaculate) and sperm concentration (×106) were found to be 6.13±0.28 and 1664.28±62.41 in Holstein Friesian breeding bulls whereas 5.26±0.17 and 2036.00±43.99 in Sahiwal bulls, respectively. The progressive motility, fast motility, slow motility and local motility were significantly (p&lt;0.05) higher in Holstein Friesian bulls than that of Sahiwal bulls both before and after freezing. Different motility patterns such as progressive, fast, slow, local and immotile motility were found to be 89.22±0.45%, 75.97±0.67%, 9.10±0.36%, 4.25±0.15% and 10.05±0.41% in Holstein Friesian bulls whereas 84.98±0.89%, 65.99±0.92%, 11.88±0.37%, 5.65±0.19% and 15.69±0.54% in Sahiwal breeding bulls, respectively before freezing. On the other hand, after freezing, the progressive, fast, slow, local and immotile spermatozoa were found to be 68.19±0.46%, 53.39±2.01%, 9.21±0.46%, 8.47±0.72% and 26.76±2.01% in Holstein Friesian bulls whereas 56.54±0.25%, 42.52±0.67%, 11.11±0.55%, 13.04±1.24% and 30.45±1.58% in Sahiwal bulls, respectively. Non-return rate was found insignificant between the breeds and it was 64.19% in Holstein Friesian and 64.71% in Sahiwal bulls. In conclusion, frozen semen quality of Holstein Friesian pure breed is better than that of Sahiwal breeding bulls but fertility of both breeds are similar. Therefore, frozen semen of both breeds can be used in the field level for the genetic improvement of cattle.&#x0D; Asian Australas. J. Biosci. Biotechnol. 2020, 5 (1), 33-41

The Acrosome Integrity Examination of Post-thawed Spermatozoa of Several Ongole Grade Bull in Indonesia Using Giemsa Staining Method

Cement cryopreservation is a preserving technique of spermatozoa cells that the spermatozoa cells can be used in a long period of time. Frozen semen is produced by the National Artificial Insemination Center (AIC) and the Regional Artificial Insemination Center (RAIC) in Indonesia. The indicators in predicting spermatozoa fertilizing ability include individual motility and movement scores, but the indicators have not been able to accurately predict the spermatozoa fertilizing ability, Acrosome integrity of spermatozoa cells is an important indicator of the success of the fertilization. The study aimed at evaluating the quality of the acrosome integrity of Ongole Grade Bull post-thawed spermatozoa from several Artificial Insemination Centers (AIC) in Indonesia using Giemsa staining method. It used 60 straw samples of 6 PO cattle aged 6 to 8 years with healthy and normal reproductive organs obtained from 3 different Artificial Insemination Centers (AICs) in Indonesia. The spermatozoa acrosome integrity was evaluated using Trypan Blue-Giemsa (TBG) and Giemsa stainings combined with fixation using methanol. The results showed that the motility (43.60±1.65%) and the viability of the spermatozoa (55.75±1.83%) of Ongole grade bull in the AIC A was higher than those in the other AIC (P<0.05). The results of the evaluation of the acrosome integrity of the Ongole grade bull from the AIC A using the TBG staining showed that the acrosome integrity of the bull from the AIC A was higher than that of the bull from the other AIC (P<0.05) (77.00±1.52%). They also showed that the acrosome integrity of the Ongole grade bull from the AIC A evaluated using Giemsa staining was also higher than that of the bull from the other AIC (P<0.05) (76.84±1.57%). Spermatozoa acrosomes can be evaluated using TBG or Giemsa stainings. However, the TBG staining is more complex and it takes a long time for the staining to give expected results. Meanwhile, the Giemsa staining combined with fixation using methanol is a simple method and it does not take a long time for the staining to give satisfying results.

The Pregnancy Evaluation on Ongole Crossbred Cows by using Liquid Semen and Frozen Semen

Artificial Insemination using liquid semen is needed for areas where there is no liquid nitrogen and the high price of liquid Nitrogen and equipment for storing frozen semen. The purpose of this study was to determine the success rate of Artificial Insemination using Frozen semen and liquid semen on different diluents in cattle Ongole crossbred. The research method used was Field experiment design. The material used was 81 Ongole Crossbred cows. Each treatment was 27 head. Control was T0 = 27 Cows in AI use frozen semen, T1 = 27 Cows in AI uses Liquid semen with CEP-3 + 10% egg Yok diluter and T2 = 27 Cows in AI use Liquid semen with Tris Amino methan + 20% egg Yok diluter. Cows were selected by purposive sampling which was normal reproduction and showing normal signs of estrus. AI methods was Recto vaginal method with 4+ semen deposition (Uterus Position). The parameter observed were Non Return Rate and Conception rate. The pregnancy determination using the per rectal palpation method. The results showed that the value of NRR-1, NRR-2 and NRR-3 in T0 = 81,48%, 81,48% and 81,48%; of T1 = 81,48%, 81,48% and 70,37% while T2 = 96,30%, 88,89% and 81,48%. Whereas the Conception rate was at T0,T1 and T2 = 62,96%, 51,85% dan 59,26%. In conclusion, the success rate of liquid semen AI using CEP-3 + 20% egg yolk diluent and Tris Aminomethan + 20% egg yolk gave the same effect compared to AI using frozen semen.

Clinical Application of in Vitro Embryo Production in the Horse

The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor mares (historically <25%; now >50%) and in the successful culture of zygotes to the blastocyst stage in vitro (historically <10%; now >20%). It has also been established that immature oocytes can be “held” at room temperature for at least 24 hours, allowing overnight transport to a laboratory with expertise in IVEP. Moreover, because in vitro–produced embryos can be cryopreserved with no appreciable reduction in viability, they can be shipped and stored until a suitable recipient mare is available for transfer. Most importantly, in an established equine ovum pick-up intracytoplasmic sperm injection (OPU-ICSI) program, blastocyst production rates now exceed 1 per procedure, and posttransfer foaling rates exceed 50%, such that overall efficiency betters that of either embryo flushing or oocyte transfer. Moreover, OPU-ICSI can be performed year round and allows embryo production from mares with severe acquired subfertility and extremely efficient use of scarce or expensive frozen semen. Cumulatively, these factors have stimulated rapid growth in demand for IVEP among sport horse breeders.

Gene Polymorphisms in Boar Spermatozoa and Their Associations with Post-Thaw Semen Quality.

Genetic markers have been used to assess the freezability of semen. With the advancement in molecular genetic techniques, it is possible to assess the relationships between sperm functions and gene polymorphisms. In this study, variant calling analysis of RNA-Seq datasets was used to identify single nucleotide polymorphisms (SNPs) in boar spermatozoa and to explore the associations between SNPs and post-thaw semen quality. Assessment of post-thaw sperm quality characteristics showed that 21 boars were considered as having good semen freezability (GSF), while 19 boars were classified as having poor semen freezability (PSF). Variant calling demonstrated that most of the polymorphisms (67%) detected in boar spermatozoa were at the 3’-untranslated regions (3’-UTRs). Analysis of SNP abundance in various functional gene categories showed that gene ontology (GO) terms were related to response to stress, motility, metabolism, reproduction, and embryo development. Genomic DNA was isolated from sperm samples of 40 boars. Forty SNPs were selected and genotyped, and several SNPs were significantly associated with motility and membrane integrity of frozen-thawed (FT) spermatozoa. Polymorphism in SCLT1 gene was associated with significantly higher motility and plasma membrane integrity of FT spermatozoa from boars of the GSF group compared with those of the PSF group. Likewise, polymorphisms in MAP3K20, MS4A2, and ROBO1 genes were significantly associated with reduced cryo-induced lipid peroxidation and DNA damage of FT spermatozoa from boars of the GSF group. Candidate genes with significant SNP associations, including APPL1, PLBD1, FBXO16, EML5, RAB3C, OXSR1, PRICKLE1, and MAP3K20 genes, represent potential markers for post-thaw semen quality, and they might be relevant for future improvement in the selection procedure of boars for cryopreservation. The findings of this study provide evidence indicating that polymorphisms in genes expressed in spermatozoa could be considered as factors associated with post-thaw semen quality.

Lyophilized seminal plasma can improve stallion semen freezability

The aim of this study was to evaluate the effect of lyophilized seminal plasma (LSP) on stallion semen freezability. Seminal plasma from 30 stallions was lyophilized to obtain a pool of LSP. Fifteen ejaculates from five stallions were supplemented before freezing with 0 mg/mL (Control), 1.44 mg/mL (LSP1), 5.04 mg/mL (LSP2) or 8.68 mg/ mL (LSP3) of LSP. Total antioxidant capacity (TAC) of LSP was assessed using Oxygen Radical Absorbance Capacity (ORAC) assay. Post-thaw motility and kinetics, sperm viability, normal morphology and membrane integrity were evaluated. Completely randomized mixed models were fitted for data analyses. The results was analyzed based on freezability of semen samples. TAC for LSP pool was 13679.4±911.6 μmol Trolox 100/g (ORAC units). Semen supplementation with LSP1 and LSP2 showed a positive effect on post-thaw total motility and membrane integrity. Supplementation with LSP3 showed a decrease in post-thaw total and progressive motility, straight line velocity and sperm viability. For poor freezability semen samples, supplementation with LSP1 and LSP2, showed higher post-thaw total motility and membrane integrity than good freezability semen samples. In conclusion, supplementation with LSP can improve the post-thaw seminal quality of stallion semen with poor freezability.

Factors affecting the pregnancy rate of Bangladeshi ewes following Laparoscopic Artificial Insemination (LAP-AI)

The study was conducted to determine the effects of different factors on increased pregnancy rate of Bangladeshi ewes through LAP-AI. A total of 24 indigenous ewes and 4 rams were selected. Frozen semen with a concentration of 100×106 spermatozoa/ 0.25 ml straws were produced. The selected ewes were synchronized using two doses of PGF2α @ 175µg per ewe at 9 days intervals intramuscularly. Estrus was confirmed by the vasectomized ram. The LAP-AI was performed at two different times from the onset of estrus, two angles of placement on the laparoscopic cradle, and two different puncture sites. Pregnancy was confirmed by ultrasonography on days 45-50 following insemination. The pregnancy rate of ewes 2.5-4 years of age and 16-20 kg bodyweight was higher (75% vs. 41.67%) than ewes 1-2 years and 9-13 kg. However, this was not significant (p&gt;0.05). The pregnancy rate of ewes having BCS &gt;3 was significantly higher (p&lt;0.05) (78.57% vs. 30%) than BCS 2-2.5. The 20-24 hours insemination time from the onset of estrus yielded a significantly higher (p&lt;0.05) (75% vs. 25%) pregnancy rate than 12-16 hours. The pregnancy rate was higher (66.67% vs. 50%) at 45° than 30°angle of placement of ewes on the laparoscopic cradle. However, there was no significant difference (p&gt;0.05). The puncture point, 4-5 cm away from the udder resulted from a significantly higher pregnancy rate (p&lt;0.05) (83.33% vs. 33.33%) than 7-10 cm. The present study indicates that the pregnancy rates following LAP-AI using frozen semen could be increased if the significant factors are considered.

Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability

Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-β-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.