Functional Sperm Parameters and Fertility of Bull Semen Extended in Biociphos-Plus® and Triladyl®

Twenty ejaculates from five dairy AI‐bulls were used to compare, in a split‐sample experiment, the fertility [56 day‐non‐return‐rate (NRR) from more than 14000 AI) and sperm viability post‐thaw of semen diluted with an egg yolk‐ (Triladyl®) or soybean‐based (Biociphos‐Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post‐thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD‐1, SYBR‐14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD‐1). Experiment 2 (n = 10) included evaluations of the capacitation‐(CTC/EthD‐1) and acrosome status (FITC‐PSA/EthD‐1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post‐thaw (CASA) showed higher values for Biociphos‐Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos‐Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos‐Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR‐14 or the ORT‐assay. When using the CTC/EthD‐1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post‐thaw (p < 0.01) was found in Biociphos‐Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos‐Plus® when semen was incubated (38°C and 5% CO₂) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR‐spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC‐PSA‐assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC‐PSA assays. In conclusion, fertility was not affected by Biociphos‐Plus® when 15 × 10⁶ of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post‐thaw, when frozen in Biociphos‐Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.     

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability

The function of prion‐like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post‐thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim‐up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post‐swim‐up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.     

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Effect of Incorporation of Additives in Tris‐Based Egg Yolk Extender on Buffalo (Bubalus bubalis) Sperm Tyrosine Phosphorylation During Cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4‐bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris‐based egg yolk extender supplemented with additives like taurine (50 mm ) or trehalose (100 mm ) or 4‐bromophenacyl bromide (200 μm ) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen‐thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris‐based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho‐proteins using SDS–PAGE followed by immunoblotting. Monoclonal anti‐phosphotyrosine antibody (Clone pT‐154) was used as primary antibody followed by treatment with HRP‐conjugated secondary antibody. Signals were detected on X‐ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post‐thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.     

Interrelationship Between Plasma Membrane Integrity, Mitochondrial Membrane Potential and DNA Fragmentation in Cryopreserved Bovine Spermatozoa

The objective of this flow cytometric study was to examine plasma membrane integrity, mitochondrial membrane potential (MMP) and the degree of DNA fragmentation of cryopreserved bovine sperm immediately (0 h) and 3 h after thawing and to compare the results with each other and with the fertility of bulls. Cryopreserved spermatozoa from 4 consecutive ejaculates of 20 bulls were examined. Percentages of plasma membrane intact sperm (PMI) and sperm showing a high MMP (HMMP), respectively, were determined by the SYBR14/PI‐ and the JC‐1 assays. DNA fragmentation was analysed by the standard deviation of the DNA fragmentation index (SD‐DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSA^TM. The mean non‐return rate on day 56 (NRR 56) ranged from 63.7% to 78.0% (mean ± SD: 71.8% ± 3.7%). Mean values for PMI and HMMP decreased from 37.4% ± 6.8% to 31.2% ± 6.1% and from 38.8% ± 7.1% to 23.8% ± 7.7% respectively. SD‐DFI increased from 56.9% ± 8.0% to 69.0% ± 12.9% and %DFI from 6.4% ± 2.5% to 12.4% ± 5.8%. The correlation between PMI 0 h and HMMP 0 h (r = 0.95; p < 0.0001) was higher (p < 0.05) than that between PMI 3 h and HMMP 3 h (r = 0.88; p < 0.0001). %DFI 0 h was neither related to PMI 0 h nor to HMMP 0 h (p > 0.05), nor was there a correlation (p > 0.05) between DFI 3 h and PMI 3 h; but %DFI 3 h and HMMP 3 h were significantly correlated (r = ?0.31; p < 0.05). SD‐DFI and %DFI 3 h were the only parameters related to NRR 56 (r = ?0.58; p < 0.05). In conclusion, plasma membranes and mitochondria are similarly affected by the freezing and thawing process, but not during the incubation period after thawing.     

Fertility Results after Vaginal Deposition of Frozen‐Thawed Buck Semen Diluted with Two Different Extenders Using One‐ or Two‐Step Procedures

In two field trials (T1 and T2), the effect of two different extenders for buck semen was tested. Semen from six (T1) and seven (T2) bucks of the Norwegian Dairy Goat breed was diluted either in a milk‐based extender containing egg yolk (M) or in a commercially available extender without egg yolk [Andromed^® (A)]. Dilution in M was performed in a two‐step procedure including centrifugation of the ejaculates and removal of the supernatant, while dilution in A was performed in one step. During the two trials (T1 and T2) 514 and 714, does, respectively, were artificially inseminated during natural oestrus, and the farmers performed the inseminations themselves after attending an artificial insemination (AI) training course. Vaginal insemination with 200 × 10⁶ spermatozoa diluted in M resulted in a 25‐day non‐return rate (NRR) and kidding rate of 37.3% and 24.5%, respectively, while semen diluted in A resulted in 31.7% NRR and a kidding rate of 19.8% (T1). In T2, NRR and kidding rate for AI performed with semen diluted with M were 42.7% and 28.5%, respectively, while dilution in A gave 37.2% NRR and a kidding rate of 26.8%. There was no significant effect of extender in the two trials [T1:p = 0.068 (NRR), p = 0.148 (kidding rate), T2:p = 0.096 (NRR), p = 0.38 (kidding rate)], but farmer had a significant effect on the fertility parameters in both trials. In conclusion, the present studies may indicate that Andromed^® is suitable for buck semen. However, more research is necessary to confirm the results and to improve the fertility of does after vaginal AI with frozen‐thawed semen.     

Detection of quantitative trait loci affecting non-return rate in French dairy cattle

The purpose of this study was to map quantitative trait loci (QTL) influencing female fertility estimated by non-return rate (NRR) in the French dairy cattle breeds Prim'Holstein, Normande and Montbeliarde. The first step was a QTL detection study on NRR at 281 days after artificial insemination on 78 half-sib families including 4993 progeny tested bulls. In Prim'Holstein, three QTL were identified on Bos taurus chromosomes BTA01, BTA02 and BTA03 (p < 0.01), whereas one QTL was identified in Normande on BTA01 (p < 0.05). The second step aimed at confirming these three QTL and refining their location by selecting and genotyping additional microsatellite markers on a sub-sample of 41 families from the three breeds using NRR within 56, 90 and 281 days after AI. Only the three QTL initially detected in Prim'Holstein were confirmed. Moreover, the analysis of NRR within 56, 90 and 281 days after AI allowed us to distinguish two FF QTL on BTA02 in Prim'Holstein, one for NRR56 and one for NRR90. Estimated QTL variance was 18%, 14%, 11.5% and 14% of the total genetic variance, respectively, for QTL mapping to BTA01, BTA02 (NRR90 and NRR56) and BTA03.     

Sequestration of PDC‐109 Protein by Specific Antibodies and Egg Yolk Cryoprotects Bull Spermatozoa

PDC‐109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time‐ and concentration‐dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC‐109 either by anti‐PDC‐109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC‐109 protein was purified by applying two‐step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion‐exchange chromatography. After checking the Ab cross‐reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris‐glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC‐PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein‐sequestered‐semen (zona‐penetration assay) were evaluated. A significant (p < 0.05) improvement in post‐thaw SQPs as well as in non‐capacitated spermatozoa observed at pre‐freeze and post‐thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein‐mediated cryoinjury. From this study, it can be concluded that sequestration of PDC‐109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra‐low temperatures.     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

The Relationship Between Post‐Thaw Sperm DNA Integrity and Non‐Return Rate Among Norwegian Cross‐Bred Rams

With the aim of investigating the relationship between sperm DNA integrity and non‐return rate (NRR) among Norwegian cross‐bred rams, semen from 15 individuals was examined by flow cytometry. Sperm Chromatin Structure Assay (SCSA) quantifies the proportion of spermatozoa with denatured DNA after in situ acid treatment, and the four parameters % DFI, % HDS, MEAN DFI and SD DFI are all different measures of DNA denaturation and maturation. Field fertility, reported as NRR 25 days after insemination was based on all inseminations from a large‐scale breeding programme and supplied by the Norwegian Association of Sheep and Goat Farmers. From each ram, four straws from four different weeks of the breeding season were analysed, and the associations between 25‐day NRR and the mean of the four SCSA parameters were tested using a logistic regression model. The results revealed no association between fertility and % DFI or % HDS, while SD DFI and MEAN DFI showed a significant negative association with NRR. Further, the SCSA values varied significantly between ejaculates within ram among some of the rams in the study. However, no significant association was seen between these intra‐individual differences in sperm DNA integrity and NRR. In conclusion, this study suggests an association between sperm DNA integrity and NRR for rams. However, further research must be conducted to confirm these findings and determine whether sperm DNA assessments can be applied to predict ram fertility.     

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin‐like Growth Factor I,Insulin and Membrane Integrity of Bull Spermatozoa

This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin‐like growth factor I (IGF‐I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre‐freezing and post‐thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment‐by‐collection day on plasma concentrations of IGF‐I and insulin was not significant. However, mean plasma concentrations of IGF‐I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment‐by‐collection day on motility of spermatozoa was similar (p > 0.05) at pre‐freezing and post‐thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post‐thawing stage was higher (p < 0.05) in rbST‐treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF‐I and insulin, however, it did improve post‐thaw sperm membrane integrity.     

Studies on the Intracellular Ca2+ Concentration of Frozen–Thawed Dog Spermatozoa: Influence of Equex from Different Sources, Two Thawing Diluents and Post-thaw Incubation in Capacitating Conditions

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris–egg yolk extender was demonstrated to improve the longevity of frozen–thawed dog spermatozoa during in vitro incubation at 38°C. The aim of the first experiment was to compare the effects of two SDS‐containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris–egg yolk based extender, on the post‐thaw longevity of dog spermatozoa, as well as on the intracellular Ca²⁺ concentration of spermatozoa, during post‐thaw incubation at 38°C. The post‐thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca²⁺ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post‐thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca²⁺ concentration of cryopreserved dog spermatozoa during post‐thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 μg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca²⁺ content. However, after 8–10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2–4‐h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris–egg yolk based extender significantly improved the post‐thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post‐thaw sperm longevity; (c) the addition of 5 μg/ml of heparin to CCM had no significant capacitating effects on frozen–thawed dog spermatozoa.     

The Effect of Oestradiol, Progesterone and Heparin on Bovine Spermatozoa Function After Thawing

The present experiment was designed to determine the effects of various biologically active substances, such as oestradiol (OE), progesterone (P4) and heparin (Hep) alone or in combination on sperm plasma membrane scrambling, capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa. Spermatozoa were incubated for 180 min in capacitation medium supplemented with (i) 1 mug/ml OE; (ii) 1 mug/ml P4; (iii) 1 mug/ml OE and 1 mug/ml P4; (iv) 1 mug/ml OE and 5 mug/ml Hep; (v) 1 mug/ml P4 and 5 mug/ml Hep; (vi) 1 mug/ml OE, 1 mug/ml P4 and 5 mug/ml Hep. At predetermined time intervals aliquots were taken to assess sperm plasma membrane scrambling, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment was aimed to study the effects of OE, P4 and OE/P4 as potential inducers of AR in Hep-capacitated spermatozoa. Plasma membrane scrambling was assessed by a flow cytometer, using Merocyanine staining. Acrosomal status and viability of spermatozoa were evaluated under epifluorescence microscope with Ethidium homodimer-1/peanut agglutinin fluorescein isothiocyanate staining method (EthD-1/PNA-FITC). The results show that OE, P4 and a combination of OE/P4 at concentrations used did not affect sperm viability. Heparin significantly (p < 0.001) increased sperm plasma membrane scrambling of OE and P4-treated spermatozoa. P4 significantly affected the rate of sperm capacitation (p < 0.001) and AR (p < 0.05), but OE expressed membrane-stabilizing properties (p < 0.05). It can be concluded that in frozen-thawed bovine spermatozoa OE presents plasma membrane stabilizing properties that can be abolished by Hep, but not by P4. Progesterone possesses capacitating and AR-inducing properties in frozen-thawed bovine spermatozoa that can be alleviated by OE.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.     

Effect of freezing bull semen in two non‐egg yolk extenders on post‐thaw sperm quality

Traditionally, extenders for bull semen included egg yolk or milk, but recently there has been a move to avoid material of animal origin. The aim of this study was to evaluate the effects of two commercial extenders (based on soya lecithin and liposomes) on bull sperm quality after cryopreservation. Post‐thaw sperm quality was evaluated by computer‐assisted sperm analysis and flow cytometric assessment of membrane integrity, chromatin integrity, mitochondrial membrane potential, production of reactive oxygen species and tyrosine phosphorylation. Furthermore, an artificial insemination (AI) trial was conducted, and 56‐day non‐return rates were evaluated. Semen frozen in the liposome‐based extender showed similar membrane integrity and higher mitochondrial membrane potential compared to those in the soya lecithin‐based extender. Chromatin integrity and production of live H₂O₂+ reactive oxygen species were similar in both extenders. Less superoxide was produced in the samples extended with liposome‐based extender, with or without menadione stimulation. Chromatin integrity and tyrosine phosphorylation were not affected by either type of extender. No differences in 56‐day non‐return rate between extenders containing soya lecithin and liposomes were observed in the AI trial (66% ± 0.8 and 65% ± 0.8, respectively). In conclusion, the sperm quality of bull semen frozen in the two extenders that do not contain material of animal origin was similar, although the semen frozen in the liposome‐based extender had higher mitochondrial membrane potential. Either extender could be used in situations where extenders containing material of animal origin are to be avoided.     

Birth of Kids After Artificial Insemination with Sex‐Sorted,Frozen‐Thawed Goat Spermatozoa

Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.     

Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen

Twenty‐four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4–8 years were used to assess various attributes of spermatozoa influencing the zona‐binding and zona‐penetration tests. Ejaculates from each bulls were subjected to in vitro sperm‐‐zona binding and sperm‐‐zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 ± 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 ± 0.64. The average number of zona‐bound and ‐penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin‐‐nigrosin stain and hypo‐osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 ± 2.32) was significantly (p < 0.05) higher than hypo‐osmotic swelling‐Giemsa (HOS‐G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 ± 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin‐‐nigrosin (r = 0.85, p < 0.05). The HOS‐G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin‐‐nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS‐G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.