Effect of Different Packages and Freezing/Thawing Protocols on Fertility of Ram Semen

A field trial was performed in order to evaluate the effect on fertility of different straw types, freezing protocols (one- or two-step) and thawing procedures (35°C and 70°C) using frozen–thawed ram semen. A total of 791 Norwegian Crossbred ewes were artificially inseminated during natural oestrus with semen collected from nine mature and proven Norwegian Crossbred rams. A milk-based extender was used for dilution. The ewes were allocated into one of the following three groups based on the different straw types and thawing temperatures: medium straw (0.5 ml) thawed at 35°C for 20 s (Med35), medium straw thawed at 70°C for 8 s (Med70) and mini straw (0.25 ml) thawed at 35°C for 15 s (Mini35). The semen to be frozen in mini straws was re-concentrated by centrifugation. Sperm number in each insemination dose was approximately 200 × 10⁶ spermatozoa. The fertility results [as 25-day non-return rate (NRR)] for Med35, Med70 and Mini35 were 53.1%, 50.8% and 58.3%, respectively, and the lambing rates 49.8%, 46.8% and 53.8%, respectively. No significant main effects were seen for straw type/thawing temperature (p = 0.17), ram (p = 0.06) or age of the ewe (p = 0.18) on NRR or lambing rates (p = 0.19, p = 0.16 and p = 0.27, respectively). Both NRR and lambing rate differed significantly among farms (p < 0.0001).     

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.     

Extenders containing dimethylformamide associated or not with glycerol are ineffective for ovine sperm cryopreservation

The study aimed at testing the effectiveness of dimethylformamide, alone or combined with glycerol, as cryoprotectant for freezing ram semen. Ejaculates from nine rams were cryopreserved in Tris-based extenders, containing 5% of glycerol, association of dimethylformamide with glycerol, in four proportions achieving 5% of cryoprotectors in the media and pure dimethylformamide (2, 3, 4 and 5%) in replacement to glycerol. The samples were diluted to 100 × 10(6) sptz/ml and stored in 0.25-ml straws in liquid nitrogen. After thawing (37 °C for 30 s), motility was preserved better by the extender containing 5% of glycerol (p < 0.05). The extenders containing pure dimethylformamide, or more than 2% in combination with glycerol, provided sperm motilities close to zero. Plasma and acrosomal membrane integrity were preserved better (p < 0.05) in the extender containing 5% glycerol. It can be concluded that dimethylformamide, alone or combined with glycerol, has no beneficial effects on ovine semen cryopreservation.     

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.     

Comparison of Membrane‐Permeant Fluorescent Probes for Sperm Viability Assessment in the Ram

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR‐14; Hoechst‐33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR‐14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane‐affected sperm (semen treated with three cycles of freezing to ?20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma‐intact sperm determined by acridine orange and SYBR‐14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.     

Impact of 24-h Cooling Prior to Freezing on the Survival of Domestic Cat (Felis catus) Epididymal Sperm

The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris–glucose–20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5°C at a rate of 0.5°C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5°C for 24 h (cooled group) and after freezing–thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5°C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze–thaw process.     

Effect of Catalase and Superoxide Dismutase on Motility, Viability and Acrosomal Integrity of Frozen-Thawed Cat Spermatozoa

The aim of this study was to evaluate the effect of antioxidant catalase (CAT) and superoxide dismutase (SOD) in semen extender on motility, viability and acrosomal integrity of frozen-thawed cat spermatozoa. Semen was collected by using an artificial vagina from five domestic cats (two ejaculates/cat). Spermatozoa were diluted in egg yolk Ttris-fructose citrate solution (EYT-FC) without glycerol and cooled at 4°C for 1 h, then diluted further with EYT-FC with glycerol (7% final concentration) and 400 IU/ml of CAT (treatment 1) or SOD (treatment 2) or without antioxidants (control). Before freezing using a styrofoam box, diluted spermatozoa filled in 0.25-ml straws were equilibrated for 1 h at 4°C. After thawing, spermatozoa were assessed for motility, viability and acrosomal integrity. Cryopreservation significantly impaired sperm motility, viability and acrosomal integrity (p   <   0.05). However, motility, viability and acrosomal integrity of frozen-thawed cat spermatozoa in the EYT-FC with CAT, SOD and without the antioxidants were not significantly different. The average percentages of spermatozoa motility after thawing compared between control, treatment 1 and treatment 2 group were 43.5 ± 3.2, 42 ± 4.1 and 38 ± 4.5; for viability: 44.8 ± 3.5, 50.6 ± 5.7 and 47.1 ± 4.1 and for acrosomal integrity: 45 ± 3.5, 44.9 ± 3.4 and 44.4 ± 3.3, respectively. In conclusion, adding CAT and SOD to EYT-FC did not improve motility, viability and acrosomal integrity in cryopreserved cat spermatozoa.     

Three Fluorescence Methods for Assessing Boar Sperm Viability

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This study compares three fluorescence methods for the evaluation of boar sperm viability and examines the interrelationships between fluorescence viability and motility of liquid semen, stored for 7 days, and fertility. One artificial insemination dose from each of 102 boars was stored for 7 days at 20°C. Plasma membrane integrity was evaluated in three different ways. First, a combination of two fluorophore probes, calcein acetylmethyl ester and ethidium homodimer-1 (EH), was used to stain samples for fluorescence microscopic analysis by making semen smears. Secondly, semen samples were stained with EH with and without the addition of a detergent (ATP-releasing reagent), and fluorescence intensities were measured with a fluorometer. Thirdly, fluorometric evaluation was carried out in the same pattern as with EH, by means of a more permeable, less specific DNA fluorochrome (more interference with RNA), Hoechst 33258. Computer-assisted motility assessment gave the values for total, rapid and progressive motility and path velocity in semen stored for 7 days. Fertility of the boars was determined by the nonreturn (NR%) rate within 60 days of first inseminations and litter size of multiparous farrowings. The results showed that all three fluorescence methods were strongly intercorrelated. All plasma membrane integrity parameters correlated significantly with motility parameters and with fertility parameters (NR% and litter size of multiparous farrowings), but motility parameters did not correlate with NR%. It seems that fluorometric measurement could prove useful for plasma membrane integrity studies in liquid boar semen. Use of the objective and fast fluorometer-based viability assay is thus suitable for several applications in sperm studies.     

Cryopreservation of Llama (Lama glama) Semen

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In order to test two extenders, and the effect of the addition of a surfactant and different freezing rates for cryopreservation of llama semen, the motility (MOT) and the integrity of acrosomes (NA) of 11 frozen ejaculates, collected with artificial vaginas from three llama males, were recorded. According to a 2 × 2 × 2 factorial design, the semen had been split and diluted comparatively with TRIS- and EDTA-extenders prepared, respectively, with and without 0.5% Equex STM and the samples frozen simultaneously 2 cm and 10 cm above the level of liquid Nitrogen. MOT of frozen-thawed semen was significantly better (p < 0.05) with TRIS-extender, although no difference for NA was recorded. The addition of surfactant as well as the compared freezing rates had no significant effect on MOT or NA. It was concluded that TRIS-extender may be promising for further fertility trials of cryopreserved semen, but centrifugation of prediluted semen would probably be necessary to get a minimum amount of sperm into the straws used as insemination doses.     

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM^® to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM^®. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN₂) vapour approximately 3 cm above the level of LN₂ for 20 min and then were plunged into LN₂. Thawing was achieved in warm water at 50°C for 12 s and then was incubated in a 38°C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM^® was added to the freezing extender. There was no difference (p   =   0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM^® was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p   =   0.02, p   =   0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM^® (p   >   0.05). The results of these investigations suggested that Equex-STM^® exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa

Ram seminal plasma increases the fertility of frozen-thawed ram spermatozoa deposited into the cervix. The aim of the current study was to compare the effect of ram seminal plasma to that of bull seminal plasma, dog prostatic fluid, protein-free TALP TrilEq (Triladyl with 0.5 mt of Equex STM paste added to each 100 mt) and heat-treated skim milk on longevity and percentages of progressively motile and aberrantly motile frozen-thawed ram spermatozoa. Three ejaculates from each of 6 rams were extended in TrilEq, pooled and frozen in straws as a single batch per ram. One hundred and eight straws (3 straws from each ram for each fluid) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 ml of the appropriate fluid at 37 degrees C and kept at that temperature for 6 h. Motility was assessed at x200 magnification immediately (time zero) and 2, 4 and 6 h after thawing. Progressive motility decreased from each time to the next (P < 0.05) and was 39.0 % (0 h), 26.0 % (2 h), 19.6 % (4 h) and 12.6 % (6 h); SEM 1.24, n = 108 for each group. Ram seminal plasma resulted in higher progressive motility than bull seminal plasma, lower than milk, and similar to the other fluids. Ram seminal plasma resulted in lower aberrant motility than protein-free TALP and similar aberrant motility to other fluids. The effect of ram seminal plasma and dog prostatic fluid was very similar. The effect of ram seminal plasma on the fertility of frozen-thawed ram spermatozoa deposited into the cervix is not due an exceptionally beneficial effect on the motility of spermatozoa.     

Effect of Different Levels of Silymarin and Caproic Acid on Storage of Ram Semen in Liquid Form

Two experiments were designed to evaluate the effect of silymarin on stored spermatozoa using four rams. In experiment 1, silymarin was evaluated as a supplement for Tris–glucose extender. Semen samples (n = 20) were diluted with extender containing 0, 50, 100, 150 and 200 μg/ml silymarin and incubated at 5°C for 72 h. Membrane integrity, acrosome integrity, sperm viability and motility were evaluated at 72 h. Concentration of malondialdehyde (MDA) was determined after 48 h. Membrane integrity was higher in 100 μg/ml silymarin (65.2%) than control group (43.2%, p < 0.05). Acrosome integrity was highest in 100 μg/ml silymarin (71.3%, p < 0.05). Progressive motility was higher in 100 (58.5%), 150 (60.62%) and 200 μg/ml silymarin (54.7%) than control group (30.7%, p < 0.05). The highest MDA concentration was observed in control group (400 mm /10 × 10⁶ sperm; p < 0.05). The goal of experiment 2 was to determine the interaction between silymarin and caproic acid on ram stored sperm. Ejaculates (n = 20) were diluted by Tris–glucose extender, added 0 (S_?) or 100 μg/ml (S₊) silymarin and 0 (C_?) or 0.3125% (C₊) caproic acid, and thereafter, aliquots were incubated at 5°C for 72 h. Membrane integrity was lower in C_?S_? (57.6%) than C_?S₊ (73.2%), C₊S_? (80.2%) and C₊S₊ (72.1%, p > 0.05). The highest sperm viability and acrosome integrity were observed in C₊S_‐ (82.4 and 80.1%, respectively; p < 0.05). There was no difference between C_‐S₊ and C₊S₊ on sperm viability and membrane integrity, progressive motility and MDA concentration (p > 0.05). Therefore, the supplementation of extender with silymarin and caproic acid improved sperm quality and caproic acid was superior to caproic acid plus silymarin.     

Effect of Dietary Supplementation with Cod Liver Oil on Cold Shock and Freezability of Boar Semen

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The effect of dietary supplementation with cod liver oil (CLO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), on resistance to cold shock and on freezability of boar semen was investigated. Ejaculates from 29 fertile Norwegian Landrace boars, randomly divided into control (n = 15) and CLO-group (n = 14), were frozen before and after a 12 week period of daily oil supplementation. Before each freezing, semen samples were taken to determine the fatty acid composition of the spermatozoa. Docosahexaenoic acid (22 : 6n-3; DHA) was the major fatty acid in total lipids. The n-3 fatty acid DHA increased in the CLO-group from 25.5 to 32.1% at the expense of the n-6 docosapentaenoic acid (22 : 5n-6), which decreased from 11.3 to 4.2% (p < 0.0001). The concentration of these fatty acids were unchanged in the control group. There was also a significant decrease of other PUFAs in the CLO-group (p < 0.05). Eicosapentaenoic acid (20 : 5n-3) was not found in any sample. At four different steps of the preservation process (30, 15, 5°C and after freezing/thawing) both motility and acrosome integrity were assessed. No significant differences were found either within or between the groups at any of the steps. In conclusion, CLO-supplementation alters the lipid composition of the membranes of boar spermatozoa, however, this does not seem to have any beneficial effect on cold shock and freezability of boar semen.     

Zootechnical and genetic aspects of a prolific merino program

In a Prolific Merino nucleus herd of 200 ewes the ovulation rate (OR) test results obtained in 169 animals between 1988 and 1993 were compared with those of 113 ewes from the same herd in 1996. Whereas earlier the ratio of individuals showing an OR > or = 4 was only 32%, that of the group checked in 1996 was 59%. This increase could be attributed to 40 ewes, both of whose parents had proven to be homozygous carriers of the prolific gene. To develop the Prolific Merino breed, 21 Booroola Merino rams were imported from New Zealand, and mostly their frozen semen was used. Of these rams, one was not a prolific gene carrier, 8 were homozygous carriers, 10 were heterozygous carriers and two had not been identified yet. Of the 36 home-bred rams, 9 proved to be homozygous by parents, 11 heterozygous, 8 homozygous, one proved to be a non-carrier, and 7 rams and their frozen semen were to be progeny tested. Six thousand doses of frozen semen from a total of 33 animals (16 imported rams and their 17 home-bred offspring) are stored in plastic straws. Sixty-three % of this is semen reserve from rams of the FecBFecB genotype, belonging to 10 ram lines. The remaining 37% is gene reserve intended for creating homozygous ram lines. Only one ram (no. 3244) was bought for the nucleus herd, the other ram lines were introduced into the herd by assortative mating, using intrauterine insemination. The average conception rate found after 472 intrauterine inseminations was 53% with large (occasionally 10-100%) individual ram differences.     

Reduced Immunolabelling of a Porcine Oocyte Membrane Protein Reflects Reduced Fertilizability of Porcine Oocytes Following Elevated Ambient Temperature

Decreased fertility in pigs is a common occurrence during summer months. An objective of the current experiments was to evaluate if elevated ambient temperature altered the oocyte plasma membrane including potential receptors for sperm. This would potentially contribute to reduced fertilizability. Treated gilts were exposed in vivo to 32°C for 12 h per day and 20°C for the remaining 12 h per day for 7 days; control gilts were exposed to 22°C for 12 h and 20°C for the remaining 12 h each day. Cumulus–oocyte complexes were also aspirated from ovaries obtained from gilts maintained at thermoneutral ambient temperature and matured in vitro at 38.5°C or 40°C. Relative abundance of a porcine oocyte membrane protein was examined by intensity of immunolabelling of the in vivo and in vitro matured oocytes evaluated with confocal microscopy; fertilizability of the in vitro matured oocytes was evaluated in in vitro fertilization assays. Oocytes obtained from gilts exposed to elevated ambient temperature for 7 days had reduced immunolabelling compared with oocytes from control gilts (p < 0.05). Similarly, oocytes matured in vitro for 44 h at elevated ambient temperature had reduced immunolabelling and reduced fertilizability compared with oocytes matured at 38.5°C (p < 0.01 and p < 0.05). These results suggest porcine oocyte quality is reduced by elevated ambient temperature and immunolabelling of oocytes with antibodies to specific membrane proteins may be effective to evaluate some aspects of oocyte quality.     

Cryo-scanning Electron Microscopy Discloses Differences in Dehydration of Frozen Boar Semen Stored in Large Containers

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50°C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from −5°C to −100°C, namely 2°C/min, 50°C/min and 1200°C/min, the lattermost by plunging the samples into liquid nitrogen (LN₂). The samples were thereafter fractured into LN₂ and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2°C/min and 50°C/min) but not at 1200°C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.     

Freezability of Tushin Ram Semen Extended with Goat or Cow Milk Based Extenders

Cow milk is used as an extender for ram semen cryopreservation. Caseins, the major proteins of milk, appear to provide some protective effect to sperm during cryopreservation. Goat milk has unique casein structure. The aim of this study was to investigate effect of goat milk, as a main semen extender, on freezability of Tushin Ram semen. For this aim, ejaculates from four Tushin rams were collected with artificial vagina and pooled. Pooled semen was separately extended with four different extenders: TRIS based (TRIS), cow skim milk based (CSM) (10 g/100 ml), cow semi‐skim milk based (CSSM) and goat semi‐skim milk based (GSSM) extenders, containing egg yolk and glycerol. The semen was cryopreserved and stored in liquid nitrogen until examination date. After thawing (at 37°C for 1 min), sperm motility, viability, morphology, acrosome and membrane integrity (HOST) were evaluated. Although, there was not any significant differences between extenders in post‐thaw percentage of viable spermatozoa (p > 0.05), Tushin ram semen extended with GSSM or CSM extenders had significantly higher post‐thaw percentage of progressive motility (25.0% and 30.8% respectively), compared with CSSM and TRIS (7.5% and 14.1% respectively, p < 0.001). Moreover, lowest abnormality percentage of post‐thaw spermatozoa were detected in ram semen extended with GSSM (49.5%) and CSM (51.5%), compared with CSSM (65.7%) and TRIS (60.7%) (p < 0.05). Whilst the results were considered, it was concluded that goat milk based extenders may be effectively and trustfully used in cryopreservation of Tushin ram semen, instead of cow milk and Tris based extenders, as a main extender.     

Sexual Performance of Rams Sequentially Exposed to Short-tailed and Fat-tailed Ewes

The objective of the study was to compare sexual performance of pure and crossbred rams, and to evaluate whether prior exposure of rams to short-tailed females would enhance their mating ability when later exposed to fat-tailed females. Twenty-two virgin, yearling Awassi (A; n = 7), F₁ Charollais × Awassi (CA; n = 7) and F₁ Romanov × Awassi (RA; n = 8) rams were subjected to sexual performance tests on six 20-min occasions. Each ram was individually exposed to two short-tailed oestrous ewes for three 20-min occasions on three consecutive days. Following 1 day of rest, the same 3-day procedure was repeated for each ram with fat-tailed ewes. Leg kicking bout frequency increased in CA and RA rams and decreased in A rams, when they were exposed to fat-tailed compared with short-tailed ewes. No differences in anogenital sniffing were observed among rams exposed to either short-tailed or fat-tailed ewes. However, greater (p < 0.001) anogenital sniffing bouts were recorded, when rams were exposed to short-tailed females. Upon exposure to fat-tailed ewes, CA and RA rams experienced a marked increase in mounting frequency compared with a slight increase in mounting of A rams (p < 0.001). The ability of Awassi rams to raise the fat tail of Awassi ewes was greater (p < 0.001) than CA and RA rams. Mating was improved in A while declining in CA and RA, when they were exposed to fat-tailed compared with short-tailed ewes (p < 0.001). Based on the results of the current study, it seems that all yearling rams were capable of mating with short-tailed ewes, whereas only Awassi rams managed to mate with fat-tailed ewes. It appears that brief exposures of yearling crossbred rams to short-tailed ewes do not improve their mating ability when later exposed to fat-tailed ewes.     

Cryopreservation of Ram Semen in Extenders Containing Soybean Lecithin as Cryoprotectant and Hyaluronic Acid as Antioxidant

A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml^‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml^‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml^‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.