Thawed Ram Spermatozoa Research Articles

The quality of ram spermatozoa after thawing with the addition of Mn2+, Zn2+ and Cu2+ nanocitrate to cryopreservation diluent

The aim of the study was to find out the effect of adding nanocitrate of Mn, Zn and Cu to the diluent for ram spermatozoa cryopreservation on its quality and ability for fertilizing. The experiment was carried out on six clinically healthy breeder 2–4-year-old rams of the Texel breed. The received ejaculates of the rams were evaluated for the volume, sperm concentration and motility and then divided into control and experimental groups. Control sperm samples were diluted with lactose-yolk-tris-citrate-glycerin medium (LYTCGM). Nanocitrates of microelements were added to the medium in experimental samples of ram sperm in the following doses: Zn2+ and Mn2+ — 2.5, 5.0 and 7.5 μg/l, Cu2+ — 1.25, 2.5 and 3.75 μg/l. The diluted sperm was packaged in straws, equilibrated for 2.5 h and frozen. After thawing of sperm we determined motility, survival of sperm, activity of succinate dehydrogenase (SDH) and cytochrome oxidase (CO), activity of antioxidant protection enzymes superoxide dismutase (SOD), glutathione peroxidase (HPO) and catalase (CAT). A dose- dependent effect of Mn, Zn, and Cu nanocitrates upon their addition to LYTCGM was established. Addition of nanocitrates of Mn, Zn to LYTCGM at a dose of 5.0 μg/l increased sperm motility by 22.2% (P<0.05) and 26.0% (P<0.01), and sperm survival, respectively, by 12.6% on (P<0.01) and 5.9% (P<0.05) compared to the control. Nanocitrates of Mn, Zn at a dose of 5.0 μg/l as part of LYTCGM caused a probable increase in SDH (P<0.001) and CO (P<0.05–0.01), which indicates a high fertilizing ability of ram spermatozoa. Similarly, when Mn, Zn nanocitrates were added to LYTCGM at a dose of 5.0 μg/l, SOD activity decreased by 29.6% (P<0.01) and 38.8% (P<0.01) and HPO activity increased by 43.5% (P<0.01) and 39.1% (P<0.01), and CAT — by 40.0% (P<0.05) and 37.5% (P<0.05), respectively. At the same time, the addition of Cu nanocitrate to LYTCGM with an increase in the dose significantly reduces the activity, survival and fertilizing capacity of thawed ram spermatozoa, and also worsens their antioxidant protection.

Glycerol-Free Equilibration with the Addition of Glycerol Shortly before the Freezing Procedure: A Perspective Strategy for Cryopreservation of Wallachian Ram Sperm.

This study investigated the effect of glycerol added in different phases of sperm equilibration on CASA and flow cytometry parameters of thawed ram spermatozoa. Sperm was collected from adult Wallachian rams. The freezing extender was glycerol-free ANDROMED® (Minitub GmbH, Tiefenbach, Germany) supplied by 6% exogenous glycerol at different stages of the cryopreservation process. The purpose of this study was to compare two strategies of glycerol addition for sperm cryopreservation. The first strategy included the use of a glycerol-free extender for the procedure of glycerol-free equilibration and chilling, with the glycerolation of the extender by 6% glycerol shortly before sperm slow freezing (GFA). The second strategy included the use of a freezing extender already glycerolated by 6% glycerol before the equilibration and chilling of sperm and following slow freezing (GA). Sperm samples were analyzed after equilibration (but before freezing) and after thawing (at T0, T1 h, and T2 h time points). iSperm® mCASA (Aidmics Biotechnology Co., LTD., Taipei, Taiwan) was used for the evaluation of sperm kinematics. Flow cytometry was used to measure sperm viability (plasma membrane/acrosome intactness) and mitochondrial membrane potential. The obtained results significantly demonstrated that the glycerol-free equilibration with the addition of glycerol shortly before freezing is a perspective strategy for cryopreservation of Wallachian ram sperm.

Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood, however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%. The aimed of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in the kinetics and viability of frozen and thawed ram spermatozoa. Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups: control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated at 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics, plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated. Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP, μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLC showed decreased levels of superoxide anion formation (O2-), a free radical detrimental to spermatozoa (P < 0.05). The use of 2 mg of CLC reduce VAP (P < 0.05) and did not have any beneficial effect on the evaluated parameters. Discussion: Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclodextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H2O2. They obtained superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group. The superoxide anion (O2-) is a free radical formed from molecular oxygen by the addition of an electron. It is generated spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases, and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group. Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

Single layer colloid centrifugation technique improves motility, viability and chromatin integrity of ram spermatozoa after thawing

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.

Cryopreservation and egg yolk medium alter the proteome of ram spermatozoa

Cryopreservation causes significant lethal and sub-lethal damage to spermatozoa. In order to improve freezing outcomes, a comprehensive understanding of sub-lethal damage is required. Cryopreservation induced changes to sperm proteins have been investigated in several species, but few have employed currently available state of the art, data independent acquisition mass spectrometry (MS) methods. We used the SWATH LC-MS method to quantitatively profile proteomic changes to ram spermatozoa following exposure to egg yolk and cryopreservation. Egg yolk contributed 15 proteins to spermatozoa, including vitellogenins, apolipoproteins and complement component C3. Cryopreservation significantly altered the abundance of 51 proteins. Overall, 27 proteins increased (e.g. SERPINB1, FER) and 24 proteins decreased (e.g. CCT subunits, CSNK1G2, TOM1L1) in frozen thawed ram spermatozoa, compared to fresh spermatozoa. Chaperones constituted 20% of the proteins lost from spermatozoa following cryopreservation. These alterations may interfere with both normal cellular functioning and the ability of frozen thawed spermatozoa to appropriately respond to stress. This is the first study to apply SWATH mass spectrometry techniques to characterise proteins contributed by egg yolk based freezing media and to profile cryopreservation induced proteomic changes to ram spermatozoa. SignificanceThis study profiles changes to the sperm proteome induced by exposure to egg yolk based media and the process of cryopreservation, and the biological consequences are discussed.

Effect of semen washing on thawed ram spermatozoa subjected to a four hour post-thawing thermal evaluation test

This study was conducted to evaluate sperm motility and qualitative characteristics of ram spermatozoa cryopreserved in the presence or absence of seminal plasma under controlled condition and subjected to a 4h post-thawing thermal evaluation test. The aim being that exposing these frozen-thawed sperm to thermal resistance test will provide useful information on their survivability in the female genital tract towards fertilizing the ovum. Briefly, ejaculates from 5 males (5years) were collected by artificial vagina, and split into two aliquots. One aliquot was diluted (1:5) in Tris-citric acid-glucose (TCG) solution and washed twice by centrifugation at 600×g for 10min, while the other aliquot was kept unwashed. Thereafter, washed and unwashed sperm were extended in a TCG-based media containing 5% glycerol and 15% powdered egg yolk (PEY) and frozen. Frozen-thawed sperm were thawed and incubated at 37°C for 4h. Sperm motility characteristics was evaluated by CASA while plasma membrane integrity (SYBR-14/PI), acrosome integrity (PE-PNA), mitochondria activity (Mitotracker deep red) and reactive oxygen species (H2DCFDA) were analysed by flow cytometry. Sperm nucleoprotein integrity assessment was done by the determination of overall levels of sperm-head disulfide bonds via spectrophotometric analysis of free-cysteine radicals’ levels. Unwashed samples generally showed better results than washed samples as regards to sperm motility characteristics irrespective of incubation. The post-thawing incubation had a significant (P<0.05) effect on acrosome integrity and mitochondria functionality irrespective of sperm treatment. This study demonstrates that the presence of seminal plasma prior to cryopreservation was beneficial in maintaining post thawed sperm motility, and as such, could be useful for ex situ ram sperm preservation towards its use for artificial insemination.

Changes to sperm surface carbohydrates following exposure to seminal plasma and freezing

Epididymal, ejaculated and frozen–thawed spermatozoa are exceptionally different sperm types. When placed directly into the uterus all sperm types display high fertility. However, when forced to traverse the ovine cervix (following vaginal or cervical AI), epididymal and frozen–thawed ram spermatozoa display dramatically lower fertility than fresh ejaculated spermatozoa. We suggest that changes to the outer carbohydrate-rich glycoprotein coat of spermatozoa represent a potential reason for altered cervical transit ability, as this is the primary interface with the female environment. As such, we used lectin binding to investigate modifications of the carbohydrate portion of sperm glycoconjugates in response to seminal plasma exposure and freezing. Fresh and frozen ejaculated spermatozoa and fresh epididymal spermatozoa were subjected to a swim up to remove loosely associated glycoproteins and remnants of freezing diluent. Spermatozoa were stained with fluorescein isothiocyanate (FITC) conjugated lectins (LPA, PNA, WGA and ConA), specific for the sugars of interest (sialic acid, galactose, N-acetylglucosamine andmannose, respectively) and counter-stained with propidium iodide (PI). Median FITC fluorescence of the PI negative (live) population was compared amongst treatments using a restricted maximum likelihood approach. The results provide strong evidence that both exposure to seminal plasma and freezing are processes which alter the outermost surface of spermatozoa. Seminal plasma exposure significantly increased N-acetylglucosamine (WGA; p<0.001) on the sperm membrane. Conversely, freezing led to a significant decrease in available galactose (PNA; p<0.01) and N-acetylglucosamine (WGA; p<0.001) as well as increased availability of mannose (ConA; p<0.001), typically a carbohydrate located proximal to the membrane in glycoconjugates. The observed changes lend support to the theory that while exposure to seminal plasmamay orchestrate meaningful alteration of the sperm surface, the freezing process further modifies this important region, possibly to its detriment. This is the first evidence that industry standard freezing of ram semen leads to large and significant changes in selected sperm surface carbohydrates. Further research is required to fully elucidate the consequenceof these changesandestablishpossiblemeans to prevent or repair the damage to the glycoprotein coat of ram spermatozoa. Research support by AWI and NSWSMBA Trust.

Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3–7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1+ and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase− spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

Effect of different media additives on capacitation of frozen–thawed ram spermatozoa as a potential replacement for estrous sheep serum

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin–hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin–hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen–thawed ram spermatozoa. Only the medium supplemented with heparin–hypotaurine–BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin–hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

Effect of Several Antioxidants on Thawed Ram Spermatozoa Submitted to 37°C up to Four Hours

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe(2+) /ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA ®) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.

Seminal plasma proteins do not consistently improve fertility after cervical insemination of ewes with non-sorted or sex-sorted frozen - thawed ram spermatozoa

The effect of supplementation of sex-sorted and non-sorted spermatozoa with seminal plasma protein (SPP) on fertility after cervical insemination was examined in the present study. Spermatozoa were sorted into high purity X and Y chromosome-bearing spermatozoa or not sorted and then either supplemented with SPP (>10 kDa) before freezing and/or after thawing (non-sorted only) or processed without supplementation. Inseminations were performed over 2 days with ewes receiving 100 or 25 million motile non-sorted spermatozoa in the cervix or uterus, respectively, or two cervical inseminations of 3.5 million motile sorted spermatozoa. Pregnancy rates in cervically inseminated ewes were unaffected by supplementation of sorted or non-sorted spermatozoa with SPP before freezing compared with no supplementation. The effect of post-thaw supplementation of non-sorted spermatozoa with SPP on pregnancy rates after cervical insemination varied with the day of insemination (P < 0.05); fertility was similar to laparoscopic insemination on Day 1 (56.0 +/- 10.2% v. 58.6 +/- 10.1%), but not on Day 2 (23.1 +/- 7.4% v. 66.7 +/- 9.2%). In conclusion, under the conditions of the present study, SPP did not consistently improve pregnancy rates after cervical insemination with frozen-thawed ram spermatozoa. This is the first report of pregnancies (5/56 ewes inseminated) after cervical insemination with frozen-thawed sex-sorted ram spermatozoa. Although the success rate is low, the findings are encouraging because ewes inseminated with the sex-sorted spermatozoa received only 7% of the recommended dose (100 million motile) for cervical insemination of frozen-thawed spermatozoa.

Seasonal variation in the protective effect of seminal plasma on frozen–thawed ram spermatozoa

The response of ram spermatozoa to seminal plasma is highly variable, in part due to the presence of both stimulatory and inhibitory factors. The aim of this study was to assess variation in the protection of ram spermatozoa during freezing by seminal plasma. The seminal plasma variables studied were season of collection, fractionation and method of supplementation. Spermatozoa were supplemented before freezing with 4 mg of seminal plasma proteins (SPPs) per 10 8 cells, and their motility and viability were assessed during post-thaw incubation (37 °C). In Experiment 1, semen was (a) frozen with no supplementation (Control) (b) extended with a Tris-based diluent (C+TRIS), or (c) supplemented with seminal plasma collected throughout the year (in the Southern Hemisphere) and pooled for January–March, April–June, July–August and October–December, and either fractionated to produce a concentrated >10 kDa seminal plasma protein retentate (>10 kDa SPP), or kept as crude seminal plasma (CP). There was no effect of season or seminal plasma type (CP or >10 kDa SPP) on motility of spermatozoa. CP and >10 kDa SPP improved the viability of spermatozoa when collected from January–September compared to Control. Supplementation with >10 kDa SPP increased viability of spermatozoa, compared to CP, when collected from January to July. In Experiment 2, >10 kDa SPP were either added directly to the spermatozoa or included in the cryodiluent or >10 kDa SPP were not supplemented (Control). Both supplementation methods improved the motility and the proportion of viable, acrosome-intact spermatozoa but direct supplementation resulted in more viable, acrosome-intact spermatozoa compared with supplementation of the cryodiluent. These results show that supplementation of ram spermatozoa with CP, or its protein component (>10 kDa SPP), before freezing protects them from freeze–thaw damage. The protective effect is greatest when seminal plasma is collected during the breeding season, fractionated with >10 kDa filters and added directly to the spermatozoa.

Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility ( P = 0.003) were used. The males were classified as having high fertility (≥42%) and low fertility (≤41%). Male fertility was not influenced by number of inseminated ewes ( P = 0.584), insemination technician ( P = 0.156), insemination date ( P = 0.323) or farm ( P = 0.207). Thawed sperm samples were employed to assess several sperm parameters for each male: motility, acrosomal integrity, viability, membrane stability, membrane phospholipid disorder, mitochondrial membrane potential and chromatin stability. These samples were used to carry out a heterologous in vitro fertilization. In vitro-matured calf oocytes ( n = 716) were inseminated with thawed ram semen and in vitro cultured for 40 h. Overall, at thawing, variability among males respect to sperm quality was high. Despite this variability, there were not differences ( P < 0.05) between fertility groups. Yield of hybrid embryos ranged from 31 to 59% between males. There were not differences between males ( P = 0.340). However, there were differences between fertility groups (high fertility: 55%; low fertility: 39%; P = 0.020). Multiple regression analysis showed that the heterologous in vitro fertility was the only predictive parameter for in vivo male fertility. Correlation between both parameters was fair ( r 2 = 0.760; P = 0.025). These results indicate that heterologous in vitro fertilization tests can be useful to predict the fertility of ram spermatozoa using calf oocytes with intact-zona pellucida.

Birth of offspring of pre-determined sex after artificial insemination of frozen–thawed, sex-sorted and re-frozen–thawed ram spermatozoa

The fertility of ram spermatozoa cryopreserved prior to, and following, sex-sorting by flow cytometry was assessed after insemination of mature Merino ewes at a synchronised estrus. Ewes were inseminated with spermatozoa from three rams, split into four treatment groups: 50 × 10 6 motile non-sorted, frozen–thawed (Control 50), 15 × 10 6 motile non-sorted, frozen–thawed (Control 15), 15 × 10 6 motile sex-sorted, frozen–thawed (SF 15) or 15 × 10 6 motile frozen–thawed, sex-sorted, re-frozen–thawed (FSF 15) ram spermatozoa. Separation of SF 15 and FSF 15 treatments into X- and Y-chromosome-bearing populations was achieved using a high-speed sperm sorter. The percentage of ewes lambing after insemination was similar for Control 15 (36/74; 48.6%), SF 15 (35/76; 46.1%) and FSF 15 (26/72; 36.1%) groups ( P > 0.05). A higher percentage of ewes produced lambs in the Control 50 (38/70; 54.3%) than the FSF 15 group ( P < 0.05). Fifty-one of the 55 (92.7%) lambs derived from fresh, sex-sorted frozen–thawed spermatozoa were of the predicted sex, as were 41/43 (95.3%) lambs derived from frozen–thawed, sex-sorted, re-frozen–thawed spermatozoa. This study demonstrated for the first time in any species that frozen–thawed spermatozoa, after sex-sorting and a second cryopreservation step, are capable of producing offspring of the predicted sex following artificial insemination.

In vitro characteristics of fresh and frozen - thawed ram spermatozoa after sex sorting and re-freezing

The in vitro function of sex-sorted, frozen-thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen-thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen-thawed controls immediately following thawing and after incubation at 37 degrees C for 3 and 6 h. Similarly, frozen-thawed, sorted, re-frozen-thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0-6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen-thawed, sorted, re-frozen-thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen-thawed controls.

Effect of medium on the kinematics of frozen-thawed ram spermatozoa.

Cervically inseminated cryopreserved ram spermatozoa have reduced fertility due to poor mucus-penetrating ability. This effect is ameliorated by the addition of 20% (v/v) seminal plasma (SP) to the phosphate-buffered saline (PBS) thawing medium. The aims of this study were to determine whether the impaired mucus penetration was due to alterations in the sperm motility and, if so, whether these alterations were due to the SP or its viscosity, or to the medium components. To this end, artificial SP medium (ASP), a medium which supports motility but not capacitation, was compared with PBS and SP. Thawed, pooled semen from seven mature rams was layered under 1 ml each of PBS, SP and ASP and motile spermatozoa allowed to swim up (37 degrees C, 30 min). Upper regions of the overlays were harvested, and the capacitation status of the spermatozoa in each suspension determined by chlortetracycline (CTC) analysis. Sperm movement was videotaped in 300 microm chambers for both computer-aided sperm analysis assessment and manual flagellar curvature analysis. There was no effect of the culture medium on the concentration of spermatozoa recovered by swim up, nor on the proportion of motile spermatozoa. However, the spermatozoa resuspended in PBS did show changes associated with capacitation in both the CTC-binding patterns and in their movement patterns. These changes were significantly greater than those observed in spermatozoa resuspended in SP or ASP. These results indicated that the differences in sperm movement and function observed in SP medium were not due to changes in viscosity, but rather to components of the medium.

Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen-thawed sorted X- or Y-chromosome-bearing spermatozoa.

The fertilizing ability of sex-sorted frozen-thawed ram spermatozoa was assessed after insemination of mature Merino ewes at a synchronized oestrus. Ewes were inseminated into the uterus or utero-tubal junction (UTJ) with a total of 140 x 10(6) unsorted (control) or 2-4 x 10(6) sorted (X or Y) frozen-thawed ram spermatozoa 54 to 57 hours after removal of progestagen-impregnated pessaries and an injection of 400 IU of pregnant mare serum gonadotrophin (Folligon, Intervet). The spermatozoa were separated into X- and Y-chromosome-bearing spermatozoa after analysis with a modified high-speed cell sorter (SX MoFlo). The number of ewes pregnant after insemination with unsorted frozen-thawed spermatozoa was significantly higher (26/48; 54.3%) than for ewes inseminated with either X- (12/48; 25.0%) or Y-sorted spermatozoa (7/48; 14.6%) (P<0.05). Seventeen of the eighteen lambs produced by ewes inseminated with X-sorted spermatozoa were female (94.4%) and 8/8 lambs from ewes inseminated with Y-sorted spermatozoa were male (100%). The sex ratio of the lambs born to ewes inseminated with sex-sorted spermatozoa was significantly skewed from the 51.3% male and 48.7% female ratio in the control group (P<0.05). This study showed, for the first time, that lambs of predicted sex can be produced after insemination with low numbers of sex-sorted cryopreserved ram spermatozoa.