Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters,lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.     

The protective effect of egg yolk from different avian species during the cryopreservation of Karayaka ram semen

Egg yolk is one of the most widely used cryoprotective components for sperm preservation and a wide range of factors affect its action on sperm motility, viability and fertilizing ability. The aim of this experiment was to determine the effect of different species egg yolk, namely the domestic chicken (Gallus gallus domesticus), the goose (Anatidae anser), turkey (Meleagris gallopavo), duck (Anatidae anas platyrhynchos), Japanase quail (Coturmix japonica) and chucker (Alectoris chukar) on sperm quality following cryopreservation of ram semen. Ejaculates were collected using the artificial vagina from three Karayaka rams and spermatological characteristics assessed for the pooled semen. Semen samples were evaluated as split ejaculates in the trial and samples extended with a Tris-citric acid-glucose extender containing the different avian egg yolk (15%) and glycerol (5%). The semen straws were equilibrated at 4 °C for 2 h, frozen in liquid nitrogen vapour (for 15 min at ?120 °C) and stored in liquid nitrogen (?196 °C). After thawing (37 °C for 30 s), sperm motility, viability, abnormal acrosome and membrane integrity (HOST) were evaluated. Results showed chucker egg yolk to have the best cryoprotective effect in terms of the highest sperm motility (54.0%), compared to the other five avian egg yolks (p < 0.05) evaluated. Sperm frozen in chucker egg yolk also showed a higher percentage viability (59%), than sperm stored in quail and turkey egg yolk (p < 0.05). The percentage of acrosomal abnormalities after thawing was lower in the chucker egg yolk, than the other species (p < 0.05). There was no significant difference in sperm membrane integrity between the egg yolks, except for the quail (p < 0.05). Results suggest that chucker egg yolk could be used as an alternative for chicken egg yolk, in a semen extender in cryopreservation, but it warrants further evaluation in fertility trials.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze–thawing process. The aim of this study was thus to determine the effects of anti-oxidants on standard semen parameters, lipid peroxidation (LPO) and anti-oxidant activities after the freeze–thawing of ram semen. Ejaculates collected from four Akkaraman rams, were pooled and evaluated at 33 °C. Semen samples were diluted in a Tris-based extender containing the anti-oxidants glutathione (GSH) (5 mM), oxidized glutathione (GSSG) (5 mM) or cysteine (5 mM) and an extender containing no anti-oxidants (control), cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually for 20 s in a water bath (37 °C) for microscopic evaluation. The use of an extender supplemented with cysteine led to the highest (P < 0.01) post-thaw motility (61.0 ± 1.9%), compared to the other treatment groups. No significant differences were observed in viability, acrosome damage and total abnormalities, and following the hypo-osmotic swelling test (HOST), following supplementation with anti-oxidants after the thawing of the semen. Following the thawing process, the levels of malondialdehyde (MDA) did not change with the addition of anti-oxidants, compared to the control. The GSH level and glutathione peroxidase (GSH-PX) activity remained significantly higher upon the addition of GSH (3.33 ± 0.14 nmol/ml and 22.02 ± 1.27 IU/g protein) and GSSG (3.24 ± 0.08 nmol/ml and 20.17 ± 3.38 IU/g protein) compared to the other treatment (P < 0.001) groups. Only cysteine significantly elevated the activity of catalase (CAT, 842.40 ± 90.42 kU/l) following the freeze–thawing process. The Vitamin E (VitE) level was significantly higher, when compared to GSSG, cysteine and the control, when GSH (4.21 ± 0.20 mg/dl) was added to the freezing extender (P < 0.001). It could be concluded that future efforts aimed on improving the efficiency of cryopreservation of ram sperm should concentrate on the use of anti-oxidant additives. The results obtained provide a new approach to the cryopreservation of ram semen, and could positively contribute to intensive sheep production.     

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 × 10⁶ spermatozoa/mL. The semen samples were packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (?196 °C). After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.     

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris–egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P < 0.05) between glycerol and DMF with regard to subjective progressive motility (23.9 ± 2.2% vs. 16.6 ± 2.0%), objective progressive motility (3.5 ± 0.4% vs. 1.8 ± 0.3%), linearity (53.9 ± 1.6% vs. 48.1 ± 1.4%) and amplitude of lateral head (2.3 ± 0.1 vs. 2.9 ± 0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.     

Pre-incubation prior to semen processing and the subsequent effect on the quality of fresh-cooled and cryopreserved ram semen

For artificial insemination (AI) in the pig, semen is routinely maintained at room temperature for 2–4 h prior to extending—to reduce the cooling damage to sperm during cryopreservation. In the sheep industry, however, semen is diluted and cooled immediately after collection. This trial evaluated the effect of a 4 h pre-incubation period for semen at room temperature on the subsequent quality parameters of ram sperm prepared for AI. Immediately following collection, ram semen was divided in 2 aliquots—one was left undiluted for 4 h at room temperature (20 °C; pre-incubation) and the other (control) was diluted with an egg-yolk-based extender and either cooled to 5 °C (n = 8 different ejaculates) for short-term fresh conservation or cryopreserved (n = 6 different ejaculates). After 4 h at room temperature, the pre-incubated semen was then diluted and either cooled to 5 °C or cryopreserved, as was the control. Sperm motility, viability and chlortetracycline (CTC) pattern distribution of the pre-incubated semen were compared to the control. For fresh semen conserved at 5 °C, total sperm motility and the proportion of CTC pattern F sperm (referring to non-capacitated, non-acrosome reacted cells) were reduced by the 4 h incubation at room temperature, compared to the control. The effect of pre-incubation at room temperature was more evident in the cryopreserved semen in terms of total and progressive sperm motility, with the viability being reduced following pre-incubation. For the cryopreserved semen, the percentage of CTC pattern F sperm declined, while the pattern of AR sperm (referring to acrosome-reacted cells) increased, compared to the controls. In conclusion, pre-incubation of ram semen for 4 h at room temperature prior to preparation for AI is not beneficial to the subsequent functionality of the sperm. Furthermore, this pre-incubation period is more harmful to frozen-thawed than to fresh-cooled sperm.     

Protective effect of taurine,glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h (P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h (P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C (P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation

Clove bud (Syzygium aromaticum) extract was added at concentrations of 0, 35, 75, and 115 μg/ml to ovine semen extenders in order to investigate the antioxidant activities of clove bud extract and its effects on semen quality parameters after cryopreservation of ram spermatozoa. The basic extender was composed of Tris, egg yolk, and glycerol. Two other extenders were prepared by substitution of egg yolk with either LDL or egg yolk + SDS. The DPPH inhibition test was employed to assess the antioxidant activity of clove bud extract. Results showed that, compared to vitamin E, clove bud extract had a higher antioxidant activity. Better sperm motility and movement characteristics (P < 0.05) were observed in the semen diluted with medium containing egg yolk + SDS than in that containing egg yolk and LDL. Progressive motility and movement characteristics of the sperm were significantly improved (P < 0.05) by adding 35 and/or 75 μg/ml of clove bud extract to semen extenders. Sperm viability and plasma membrane integrity were also higher (P < 0.05) in the semen exposed to medium containing egg yolk + SDS and 75 μg of clove buds extract after cryopreservation processes. Higher levels of clove bud extract, however, had adverse effects on all the sperm quality parameters and significantly reduced (P < 0.05) the motility, movement parameters, viability, and plasma membrane integrity of ovine sperm. It was concluded that the clove bud extract had an antioxidant potential that makes it useful for addition to semen extenders and that the best results are obtained with a maximum clove bud extract of 75 μg/ml. Moreover, the combination of egg yolk and a detergent was found to improve sperm quality after the cooling and freeze–thawing processes.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen–thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen–thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.     

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen–thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen–thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

First pregnancies in jennies with vitrified donkey semen using a new warming method

Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 × 10⁶ sperm) in the uterine body either with vitrified or frozen semen (2 cycles/jenny). Pregnancy rates and the uterine inflammatory response (polymorphonuclear neutrophil concentration; PMN) were evaluated after artificial insemination (AI). No differences between warming in extender/water bath were found and 43 °C/10 s was better than lower temperatures in terms of total (53.8 ± 13.2%) and progressive sperm motility (41.4 ± 11.4%). No differences in PMN concentration (× 10³ PMN/ml) were found between vitrified (276.8 ± 171.6) or frozen (309.7 ± 250.7) semen after AI. However, PMN decreased faster (P < 0.05) using vitrified semen. Pregnancy rates were greater for vitrified (22%) than frozen semen (10%) but not statistically different. In conclusion, donkey sperm vitrified in straws could be directly warmed in a water bath at 43 °C/10 s, reducing the uterine inflammatory response obtained after AI and promoting positive pregnancy outcomes. These findings confirm the possibility to use vitrified semen as an alternative for AI in jennies.     

Scrotal,testicular and semen characteristics of young Boer bucks fed winter veld hay: The effect of nutritional supplementation

The aim of the work was to determine the effects of feeding winter veld hay plus a supplement on scrotal, testicular and semen characteristics in young Boer goat bucks. Fifteen Boer goat bucks (6–8 months of age), were allocated to two groups and fed ad libitum for a period of 29 days. The WH group (winter hay or control group; n = 8) received a chopped diet consisting of grass hay, predominantly Themeda triandra grass (cut during the winter) from natural pastures (veld). The WH + S group (winter hay plus supplement; n = 7) received the same diet consisting of Themeda triandra veld hay and supplemented with maize meal, molasses meal and urea. Body weights and feed intake were recorded, as well as scrotal, testicular and semen characteristics. Results indicate a detrimental effect of winter veld hay feeding on characteristics such as sperm cell abnormalities (43% in the WH group versus 24% in WH group), testicular volume (156 ml in the WH + S group versus 104 ml for WH animals) or scrotal circumference (20.7 cm in WH + S group versus 17.7 cm in WH group). It is essential to supplement the nutrition of small ruminants under winter environments, to maintain scrotal, testicular and semen characteristics, especially if the animals are to be used in the subsequent breeding season.     

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P < 0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P < 0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P < 0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P > 0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P > 0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Effects of Nigella sativa L. seed oil on abnormal semen quality in infertile men: A randomized,double-blind,placebo-controlled clinical trial

In recent years, wide utilization of herbal drugs has encouraged scientists to determine their impressive effects on health. Since Nigella sativa L. seed (N. sativa) has many uses including infertility in traditional medicine, the effects of Nigella sativa L. seed oil on abnormal semen quality in infertile men with abnormal semen quality are of interest. This study was conducted on Iranian infertile men with inclusion criteria of abnormal sperm morphology less than 30% or sperm counts below 20 × 10⁶/ml or type A and B motility less than 25% and 50% respectively. The patients in N. sativa oil group (n = 34) received 2.5 ml N. sativa oil and placebo group (n = 34) received 2.5 ml liquid paraffin two times a day orally for 2 months. At baseline and after 2 months, the sperm count, motility and morphology and semen volume, pH and round cells as primary outcomes were determined in both groups. Results showed that sperm count, motility and morphology and semen volume, pH and round cells were improved significantly in N. sativa oil treated group compared with placebo group after 2 months. It is concluded that daily intake of 5 ml N. sativa oil for two months improves abnormal semen quality in infertile men without any adverse effects.     

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing–thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7–10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen–thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 μm/s; VCL, curvilinear velocity: 50.2 μm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 μm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P < 0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P < 0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen–thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.