Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

Effects of insulin on sperm cell quality in ram semen cooled at 5°C

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (μm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.

Improving Sperm Cryopreservation With Type III Antifreeze Protein: Proteomic Profiling of Cynomolgus Macaque (Macaca fascicularis) Sperm

Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p &lt; 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo–AFP) (p &lt; 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 μg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.

CRYOPRESERVATION OF ALPACA SPERMATOZOA OBTAINED VIA POST COPULA IN A TRIS EXTENDER WITH EGG YOLK FROM THREE AVIAN SPECIES.

The objective of this study was to evaluate the cryopreservation of alpaca spermatozoa obtained via post copula in a Tris extender with egg yolk from three avian species. Forty samples of eight alpacas were collected by the post-copula method. After the collection, we proceeded to evaluate sperm volume, color, motility and concentration. The 25 samples with volume 1 ml and total motility 60% were mixed to form pool (5 samples/pool), divided into three aliquots and diluted in Tris-base with 20% egg yolk from three avian species (hen, quail, paw). These diluted samples were refrigerated for 1,5 h at 5 °C. Once this temperature was reached, the 5% glycerol basic dilutor was added, balanced for 20 min, packed in 0,5 mL straws and frozen in liquid nitrogen vapours for 20 min. The thawed samples were evaluated at different incubation times at 37 °C: 0; 1,5 and 3 h. All parameters of fresh and thawed sperm quality were analyzed using the GLM procedure (ANOVA). The samples collected (fresh) showed a motility of 69,1%, viability of 82,8%, membrane functionality of 77,9% and acrosomal integrity of 85,8%. After the cooling process, no differences were observed between the different egg yolk when comparing the sperm characteristics evaluated (p>0,05). At thawing, motility and acrosomal integrity were superior (p<0,05) when hen and quail were used compared to paw egg yolk. At 1,5 and 3 h of incubation, motility and acrosomal integrity were superior (p<0,05) in the samples with hen and quail with respect to paw. In conclusion, the use of hen and quail provided better cryoprotective action than paw egg yolk in cryopreserved alpaca sperm and incubated at 37°C for 3 h

SNP Identification in Sperm Associated Antigen 11B Gene and Its Association with Sperm Quality Traits in Murrah Bulls

Background: The beta-defensin family antigens present on sperm surface suggestively play pivotal role in sperm fertility by aiding in various steps of sperm maturation, motility, capacitation, immune recognition and sperm-oocyte interaction in female tract. It is imperative to explore genetic polymorphisms to build a better understanding of individual variation in male fertility. The experiment was designed to identify DNA polymorphism in Sperm Associated Antigen 11B (SPAG11B) gene and to analyze association between genetic variants with sperm quality traits in buffalo bulls (Murrah) in ICAR-National Dairy Research Institute, Karnal. Methods: Genomic DNA was extracted from hundred and thirty Murrah bulls. A 395 base pair region covering partial Intron 2, Exon 3 and Partial Intron 3 of bovine SPAG11 gene was amplified and genotyped using PCR-RFLP method. The PCR amplified product was purified, sequenced and further ClustalW analysis was done to align edited sequence with reported Bos taurus sequence (AC_000158.1). Gene and genotype frequencies, effective number of alleles, level of heterozygosity and polymorphic information content of various genotypes were estimated by standard procedure. Seminal parameters (Post thaw sperm motility, sperm viability, acrosomal integrity, HOST and abnormality) were estimated and statistical analysis was carried out. Result: A novel SNP (G greater than A substitution) at 2266 base of the SPAG11B gene was identified by sequencing. The fragment of SPAG11 gene containing 395 base pairs was amplified using PCR and digested with restriction enzyme i.e. MunI which showed three distinct genotypes viz., AA (266 bp and 107 bp fragment), AG (307 bp, 266 bp and 107 bp) and GG (373 bp fragment). Least squares means of seminal parameters for the SNP was estimated and compared after correction for non-genetic factors. Between genotypes, significant differences were found only for acrosomal integrity and was highest in AG (74.22±0.72) followed by AA (72.6±0.89) and GG (71.12±0.97), respectively. The identified novel SNP of SPAG11 gene showed significant association with acrosome integrity with genotype AG being superior to other genotypes. However, an association cannot be established with other seminal parameters with this SNP, further studies are required in order to validate the impact of g.2266G greater than A on sperm quality traits in a large population.

Partial Deoxygenation of Semen Extender Minimizes Post-thaw Damages and Improves Freezability of Crossbred Bull Spermatozoa

Background: Oxidative stress occurs when oxygen or oxygen derived oxidants exceed antioxidants and become responsible for poor post-thaw semen quality during the cryopreservation process. Therefore, the present study was designed to investigate the effects of dissolved oxygen (DO) levels (4, 6 and 8 ppm) in semen extender on freezability of crossbred bull spermatozoa. As the gap between 4 and 8 ppm existed in earlier studies, DO of 6 ppm in semen extender was further standardized and the effects were compared with other respective groups to contemplate any improvement in post-thaw semen quality.Methods: For the experiment, Tris-egg Yolk-Glycerol (TYG) extender was partially deoxygenated by nitrogen gassing @ 2-3 bubbles/sec at 34°C for 0, 16, 12 and 9 minutes to obtain DO levels of 11.7 ppm (Group-I/control), 4 ppm (Group-II), 6 ppm (Group-III) and 8 ppm (Group IV), respectively and collected semen samples were diluted with these extender groups to have 80×106 spermatozoa/ ml of the extender. Semen samples were evaluated for individual progressive motility (IPM), lipid peroxidation (LPO), total antioxidant capacity (TAC), plasma membrane integrity, acrosomal integrity and apoptotic changes at different stages of cryopreservation.Result: At the post-thaw stage, progressive motility was greater (p less than 0.05) in Group II compared to Group I and the least reduction from the post-dilution to the post-thaw stage was observed in Group II. In comparison to Group I, Groups II, III and IV showed lesser (p less than 0.05) MDA production with Group II having greater (p less than 0.05) TAC concentration than other groups at the post-thaw stage. A declining trend was observed in membrane integrity as DO levels increased from 4 ppm to 11.7 ppm. Acrosomal integrity did not differ among treatment groups, but, found to be higher (p less than 0.05) than the control group. Per cent viable spermatozoa was greater (p less than 0.05) in Group II than Group I and vice versa for necrotic spermatozoa as assessed by Annexin VFITC/PI staining. In conclusion, reducing the DO level to 4 ppm before cryopreservation improved the freezability by reducing oxidative stress and apoptotic changes while, above 4 ppm tended to lower it. An appreciable improvement in freezability can be seen at 6 ppm of DO, but, not up to that extent as observed at 4 ppm.

Effect of Two Cryopreservation Methods on Seminal Quality and Pregnancy Rate in Alpacas Induced to Ovulation with Seminal Plasma

The objective was to evaluate the effect of two methods of freezing on seminal quality and effect of seminal plasma as an inducer of ovulation in pregnancy percentages in inseminated alpacas. The semen collection was made post copula. After the collection of the ejaculates, motility and volume were assessed. The ejaculates with volume (≥1 ml) and motility (≥ 60%) were mixed (pool), 80 % was used (3 samples/pool) and 20 % (2 samples/pool) during the experiment. Then and diluted in Tris-base with 20% egg yolk. The samples were cooled 1.5 hours at 5 °C. At that temperature it was combined with the basic dilutor plus glycerol, obtaining a final concentration of 5% glycerol, then they were packed in 0.5 mL (13 x 106 spem/straw) straws to be frozen by horizontal and vertical methods. The seminal (semen + vaginal fluid) quality analysis was performed fresh and after cooling and thawing. Insemination was performed in two groups with thawed semen from two straws of the vertical method, the first group 20 females induced to ovulate with GnRH analog, the second group 20 females induced to ovulate with seminal plasma. When comparing, the results obtained of motility, viability, HOST and acrosomal integrity of fresh sperm, after the freezing process, decreased (p<0.05) compared to fresh and refrigerated samples. On the other hand, when comparing the freezing methods, the sperm values frozen by the vertical method were higher than those obtained by the horizontal method, with (p<0.05) in motility and HOST without (p>0.05) in acrosomal integrity and viability. The vertical semen freezing method can replace the horizontal method to obtain pregnancy.

Does resveratrol preserve the integrity of the sperm membrane in goats after thawing?

This study aimed to determine the effects of resveratrol in the trisaminomethane (TRIS)-egg yolk extender and its optimal inclusion level for goat semen cryopreservation. Five ejaculates of three Anglo Nubian goats were used, each divided into four 200 µL aliquots for use in four treatments: 0.00 (control), 0.04, 0.08 and 0.12 mg mL-1 resveratrol in the TRIS-egg yolk extender. We evaluated progressive sperm motility and sperm vigor post-dilution, post-cooling, and post thawing; membrane integrity (HOST); and acrosomal integrity and performed a slow thermoresistance test (STT). The data were submitted to a regression analysis at a 5% probability. There was no difference in progressive motility or sperm vigor in the post-dilution (89.5, 89.0, 88.7 and 88.3, and 4.9, 5.0, 4.9, and 4.9) or post-cooling (81.0, 82.0, 83.0, and 78.3; and 4.3, 4.3, 4.2, and 4.2) experiments (P > 0.05), or in the complementary acrosomal integrity test (42.0, 47.4, 42.2 and 38.2) (P > 0.05). However, the motility and vigor parameters decreased linearly in the post-thaw phase, as well as during the 2 hours of incubation on STT (P < 0.05). These factors increased quadratically when resveratrol was added to HOST, to an optimal level of 0.039 mg mL-1 resveratrol for a plasma membrane integrity of 52.55% (P < 0.05). The inclusion of resveratrol was effective in maintaining sperm viability; in particular, it was effective in maintaining plasmatic membrane integrity during the cryopreservation process up to 0.039 mg mL-1, meaning that it could be an alternative to conventionally used seminal extenders in goats.

The Effect of Penicillin on the Vitality of Bull Spermatozoa

Antibiotic supplementation into semen extenders is an important way to control several microorganisms that can affect semen quality by their presence. The objective of the present work is to estimate the effect of two different concentrations (300 µg/mL and 600 µg/mL) of penicillin on the selected quality parameters of spermatozoa collected from bulls (motility, mitochondrial activity, acrosome integrity and membrane integrity) after 0, 2 and 24 h of in vitro culture. Sperm motion was examined using HTM IVOS computer-aided sperm analysis (CASA), cell viability was assessed with the metabolic activity (MTT) assay. The acrosomal integrity was evaluated following the fast green – rose bengal staining protocol and the eosin – nigrosin staining method was used to assess the functional integrity of the sperm membrane. Our results indicate that penicillin at lower amount significantly (p>0.05) decreased the sperm motility, mitochondrial activity and membrane integrity after 24 h of in vitro culture. Supplementation of higher doses of this substance led to a significant decrease of the sperm motion during 0, 2 (p>0.05) as well as after 24 h (p>0.01), of the viability after 2 h (p>0.05) and 24 h (p>0.01), of the acrosomal integrity after 24 h (p>0.05) and of the membrane integrity at 24 h (p>0.001) too. We can consider, that the effect of penicillin addition to bovine spermatozoa during in vitro incubation is time and dose dependent.

Fresh and Post-Thaw Seminal Attributes of Jafarabadi Buffalo Bulls

The study was conducted on four Jafarabadi breeding bulls, 5-6 years old to know the fresh and post-thawed seminal characteristics based on total of 192 semen ejaculates evaluated and cryopreserved over one year period. The mean values of fresh neat seminal characteristics of Jafarabadi bulls, viz., ejaculate volume (ml), colour/density (score), sperm concentration (million/ml), mass activity (score), initial motility (%), live sperm (%), abnormal sperm (%), HOS reactive sperm (%) and acrosomal integrity (%) were 5.19±0.18, 2.38±0.10, 1253.36±24.75, 3.73±0.05, 80.31±0.05, 86.20±0.64, 5.00±0.40, 85.75±0.43 and 93.56±0.56, respectively, whereas the mean post-thawed sperm characteristics, viz., progressive sperm motility, live sperm (%), abnormal sperm (%), HOS reactive sperm (%), acrosomal integrity (%) and first insemination conception rate (%) observed were 57.60±0.36, 66.34±0.53, 8.85±0.33, 56.97±0.46, 75.26±0.17 and 44.63±0.14, respectively. The semen quality of fresh and post-thawed samples observed was within normal limit for use in breeding program with satisfactory first insemination conception rate.