Saturated,omega‐6 and omega‐3 dietary fatty acid effects on the characteristics of fresh,frozen–thawed semen and blood parameters in rams

The aim of this study was to investigate the effects of several dietary fatty acids (FAs) on semen quality and blood parameters in rams. We gave diet‐supplemented treatments (35 g day^?1 ram^?1) by C16:0 (palm oil), C18:2 [sunflower oil (SO)] and an n‐3 source [fish oil (FO)] to 12 rams, who were fed for 15 weeks during their breeding season. Semen was collected once per week. Semen samples were extended with Tris‐based cryoprotective diluents, then cooled to 5 °C and stored in liquid nitrogen. Positive responses were seen with FO after 4 weeks. The mean prefreezing semen characteristics improved with the intake of FO (P < 0.05). Interestingly, maximum sperm output in FO was achieved 7.5 × 10⁹ when compared to palm oil 5.3 × 10⁹. Rams that received FO had the highest total testosterone concentrations (11.3 ng ml^?1 for FO, 10.8 ng ml^?1 for SO and 10.2 ng ml^?1 for palm oil) during the experiment (P < 0.05). FO also improved the rams' sperm characteristics after thawing (P < 0.05). Although C16:0 is a major saturated FA in ram sperm and all rams have been fed isoenergetic rations, the unique FAs of FO improved fresh semen quality and freezing ability compared to other oils.     

The micronutrient supplements,zinc sulphate and folic acid,did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty‐three OAT men participated in a 16‐week intervention randomised, double‐blind clinical trial with daily treatment of folic acid (5 mg day^?1) and zinc sulphate (220 mg day^?1), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A₃ staining; and semen and blood folate, zinc, B₁₂, total antioxidant capacity ( TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10⁶ ml^?1) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.     

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk‐based extender with Trolox supplementation

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml^?1] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post‐thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml^?1 Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml^?1 groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml^?1 ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.     

Reproductive and toxic effects of methanol extract of Alchornea cordifolia leaf in male rats

Alchornea cordifolia leaf is traditionally used for the treatment of venereal diseases and for the enhancement of fertility throughout its area of distribution in Africa. The effect of oral administration of the methanol extract of the leaf was evaluated on some reproductive and haematological parameters of male rats at 0 (control group), 100, 200, 400, 800 and 1600 mg kg^?1. The toxicity study revealed nonsignificant alterations (P > 0.05) in the values of total and differential white blood cell count, but the erythrocyte count, packed cell volume, haemoglobin concentration and haematometric indices were significantly decreased (P < 0.05) at 1600 mg kg^?1 dose. Markers of hepatic damage (aspartate aminotransferase and alanine aminotransferase) and renal damage (urea and creatinine) were significantly elevated (P < 0.05) at 800 and 1600 mg kg^?1. The bioactivity (reproductive) study revealed significant increases (P < 0.05) in testicular weight, sperm count and motility, and serum testosterone levels, at the 200 and 400 mg kg^?1. The study concludes that the extract of Alchornea cordifolia leaves has toxic potential at 800 mg kg^?1 and 1600 mg kg^?1 doses, but is safe and has beneficial effects on male reproduction when used at doses equal to or lower than 400 mg kg^?1.     

Spermicidal activity of Indian seaweeds: an in vitro study

Contraceptive properties of seaweeds are still stands as lacuna; in this context, the screening of in vitro male contraceptive properties of crude ethanolic extract of Indian seaweeds against normal human sperm is carried out. In total, twelve seaweeds were screened for in vitro spermicidal activity. Among these twelve seaweeds, Halimeda gracilis showed 100% inhibition of human spermatozoa at 10 mg ml^?1 concentration in 20 s and its EC₅₀ value was 2.05 mg ml^?1 in 20 s. Further, dose‐ and time‐dependent spermicidal assay revealed that the sperm was completely immobilised for 20 s. Plasma membrane of sperm was damaged due to the exposure of H. gracilis extract. MTT assay with H. gracilis extract showed 88.5% of cytotoxic incidence. H. gracilis extract tested for cytotoxicity against Artemia salina recorded LC₅₀ value of 34.8 μg ml^?1. Phytochemical analysis of H. gracilis extract evidenced the presence of alkaloids, flavonoids, proteins and sugars. Results of this study clearly inferred that the synergistic effect of active principles reside within the H. gracilis extract had shown better contraceptive activity.     

Prostate‐specific targeting of the aqueous root extract of Croton membranaceus in experimental animals

Croton membranaceus Müll.Arg. (Euphorbiaceae) is used for benign prostate hyperplasia (BPH) treatment. The study aimed at investigating organs that the aqueous root extracts of C. membranaceus (CMARE) target, which is absent in literature. Twenty‐four male Sprague‐Dawley rats (100–140 g) were randomly divided into 4 groups. Group 1, the control group received distilled water. Groups 2, 3 and 4 received 30, 150 and 300 mg kg^?1 b.wt CMARE respectively (oral gavage). Rats fed 90 days the standard chow diet ad libitum. Upon sacrifice, major organs were histologically examined and serum prostate‐specific antigen (PSA) biochemically determined. Only the prostate was abnormal. Histologically, H&E staining revealed thickness and infoldings of the epithelial cells shrinking with increasing dose. The 30 mg kg^?1 group showed low columnar or flattened epithelium cells, whereas the columnar epithelium infoldings of the 150 mg kg^?1 b.wt and 300 mg kg^?1 b.wt groups were virtually nonexistent. The acini of the control, 30 mg kg^?1 b.wt group and the 150 mg kg^?1 b.wt groups showed clear pinkish secretion. However, secretion of the high‐dose group appeared light pink in colour and the stroma cells appeared much darker than all the treated and control group. C. membranaceus targets the prostate with significant PSA reduction (P < 0.01).     

Relevance of seminal plasma nitric oxide levels and the efficacy of SSRI treatment on lifelong premature ejaculation

The aim of this study was to determine the relevance of seminal plasma nitric oxide (NO) levels and the efficacy of selective serotonin reuptake inhibitor (SSRI) treatment on premature ejaculation. A total of 16 men (aged 32.18 ± 3.32) with lifelong premature ejaculation [intravaginal ejaculation latency time (IELT) <1 min] and 11 healthy men (control group) were included in this study. The healthy men formed Group 1, and the patients were randomly categorised into two groups. Group 2 patients received 20 mg day^?1 of paroxetine, and Group 3 patients received 50 mg day^?1 of sertraline for 4 weeks. Baseline and post‐treatment findings were compared among the three groups. Mean baseline seminal NO levels in men with premature ejaculation were significantly higher than in the healthy control group (32.24 ± 5.61 μm  l^?1 versus 19.71 ± 3.50 μm  l^?1, respectively) (P < 0.001). There was no significant difference between the sertraline and paroxetine groups in terms of IIEF scores, IELT scores and NO levels. At the end of the first month, the mean IELT scores of the paroxetine and sertraline groups showed a significant improvement compared with the baseline values (P < 0.001). After treatment with paroxetine and sertraline, NO levels dec‐reased from baseline. Our study indicates that premature ejaculation is significantly related with a higher level of seminal NO. Baseline seminal plasma NO values obtained in patients with premature ejaculation were significantly higher than in the healthy control group. After treatment with SSRIs, decreased seminal NO may retard ejaculation. Further studies are needed to confirm this suggestion and the role of NO in the pathophysiology and treatment of premature ejaculation.     

Organic selenium supplementation increases PHGPx but does not improve viability in chilled boar semen

This study evaluated the effects of dietary organic selenium (Se) on viability of chilled boar semen. Twelve boars were divided into three groups: control (CON), 0.3 mg kg^?1 sodium selenite; inorganic (INO), 0.5 mg kg^?1 sodium selenite and organic (ORG), 0.5 mg kg^?1 Se yeast. The experiment was conducted within 10 weeks, and analysis was performed fortnightly, in storage semen by 72 h. No effect was observed on motility; however, straightness and linearity percentages were higher (P < 0.05) in the animals receiving CON diet compared with INO group. Percentages of cells with both plasma and acrosomal intact membranes, lipidic membrane peroxidation and mitochondrial membrane potential were similar on all treatments. Animals receiving CON diet presented higher (P < 0.05) values of ATP when compared with INO group. The PHGPx was higher (P < 0.05) in animals that received ORG in comparison with INO group. In conclusion, organic selenium supplementation increases PHGPx but does not improve chilled semen viability in 72 h.     

In vitro effects of nonylphenol on motility,mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml^?1 for 1, 2, 3 or 4 h. Computer‐assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml^?1 was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml^?1) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml^?1 NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose‐dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml^?1 NP for boar spermatozoa and 10 μg ml^?1 NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.     

Comparison of the semen analysis results obtained from two branded computer‐aided sperm analysis systems

The study evaluated the comparability of two branded computer‐aided sperm analysis (CASA) systems commonly used in andrology laboratories in China. The same semen sample was analysed using two branded CASA systems (WLJY‐9000 and CFT‐9200) by one well‐trained technician. Results of semen analysis obtained from two branded CASA systems were then compared. The accuracy of counting results of CASA systems was evaluated using latex bead solutions with known concentrations of (35 ± 5) × 10⁶ ml^?1 and (18 ± 2.5) × 10⁶ ml^?1. There were significant differences in all parameters (P < 0.01) except for LIN and WOB. The counting results of CFT‐9200 were close to the standard solutions [(38.86 ± 3.79) × 10⁶ ml^?1 and (19.03 ± 1.99) × 10⁶ ml^?1], while those of WLJY‐9000 were underestimated [(28.53 ± 2.06) × 10⁶ ml^?1 and (14.62 ± 0.95) × 10⁶ ml^?1]. But the coefficient of variation of WLJY‐9000 was lower than that of CFT‐9200 (7.22%, 6.50% vs. 9.82%, 10.46%). It is concluded that factors such as parameter settings and evaluation algorithms could significantly affect the results obtained from these two branded CASA systems. Great attention should also be paid to the quality control in semen analysis with CASA.     

Cryoprotective effect of l‐carnitine on motility,vitality and DNA oxidation of human spermatozoa

Successful cryopreservation for human spermatozoa markedly influences the reproductive outcomes of assisted reproductive technologies. But in spite of its usefulness, cryopreservation significantly decreases sperm quality. l ‐carnitine has been found to improve the quality of spermatozoa in selected cases with male infertility. Here, we examined the efficacy of l ‐carnitine in improving sperm motility and vitality and reducing sperm DNA oxidation during cryopreservation. Semen samples from infertile patients (n = 22) were collected and analysed. Cryopreservation medium supplemented with l ‐carnitine was mixed with the semen at a ratio of 1 : 1 (v/v). The final l ‐carnitine concentration in each cryovial was 0.5 mg ml^?1 per 5 × 10⁶ cell ml^?1. Controls were cryopreserved without addition of l ‐carnitine. After 24 h of cryopreservation, thawed sperm samples were analysed for motility, vitality and DNA oxidation. Sperm vitality was assessed by the eosin–nigrosin test, while sperm DNA oxidation was measured by flow cytometry. Addition of l ‐carnitine significantly improved sperm motility and vitality (P < 0.05) compared with the control. The flow cytometry experiment showed no statistical difference (P > 0.05) in the levels of DNA oxidation between samples and controls. In conclusion, l ‐carnitine improves human sperm motility and vitality, but has no effect on sperm DNA oxidation after cryopreservation.     

Sperm abnormalities induced by pre‐pubertal exposure to cyclophosphamide are effectively mitigated by Moringa oleifera leaf extract

Moringa oleifera L. is a medicinal plant with potential antioxidant property. This study was aimed at investigating the chemoprotective effect of Moringa oleifera leaf extract (MOE) on cyclophosphamide (CP)‐induced testicular toxicity. Two‐week‐old male Swiss albino mice were intraperitoneally injected with phosphate‐buffered saline, 50 mg kg^?1 of CP and 25 mg kg^?1 of MOE. In combination treatment, mice were injected with 25 mg kg^?1 of MOE 24 h prior to CP injection, 24 h prior and post‐CP injection and 24 h post‐CP injection for 5 consecutive days (10 mg kg^?1). Six weeks later, mice were sacrificed to assess epididymal sperm parameters. MOE alone did not have any significant effect on sperm parameters. However, acute injection of CP resulted in significant decline in motility (P < 0.001), increase in head abnormality (P < 0.01) and DNA damage (P < 0.05). Combining MOE with CP increased the sperm density, motility and reduced head defect and DNA damage, irrespective of the schedule and dosage of MOE. Administration of MOE prior to CP significantly elevated the level of superoxide dismutase and catalase with concomitant decrease in lipid peroxidation in the testicular tissue. In conclusion, MOE may have potential benefit in reducing the loss of male gonadal function following chemotherapy.     

Gallic acid protects against cyclophosphamide‐induced toxicity in testis and epididymis of rats

The protective role of gallic acid (GA) on reproductive toxicity induced by cyclophosphamide (CPA), an antineoplastic drug, was investigated in male Wistar rats. Sixty rats were grouped into 10 rats per group. Group 1 (control) received distilled water. Rats in groups 2 and 3 received GA alone at 60 and 120 mg kg^?1 for 14 consecutive days, respectively. Group 4 received a single intraperitoneal dose of CPA at 200 mg kg^?1 on day 1. Groups 5 and 6 received a single dose of CPA (200 mg kg^?1) intraperitoneally on day 1 followed by treatment with GA at 60 and 120 mg kg^?1 for 14 consecutive days, respectively. In testes and epididymis of the treated rats, CPA administration resulted in significant elevation (P < 0.05) in malondialdehyde (MDA), nitrite and hydrogen peroxide levels. There was a significant decrease in the activities of superoxide dismutase and glutathione‐S‐transferase. Furthermore, there were significant reductions in plasma luteinising hormone (LH), follicle stimulation hormone (FSH) and testosterone levels, which were accompanied by significant decrease in sperm motility and viability in CPA‐treated rats. Histological examination revealed marked testicular and epididymal atrophy in CPA alone treated rats and these aberrations were reversed by GA. In conclusion, GA has capacity to protect against reproductive toxicity induced by cyclophosphamide.     

Additional deleterious effects of alcohol consumption on sperm parameters and DNA integrity in diabetic mice

The aim of this study was to survey the impact of alcohol consumption on sperm parameters and DNA integrity in experimentally induced diabetic mice. A total of 32 adult male mice were divided into four groups: mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg kg^?1, single dose, intraperitoneal) and basal diet, group 3 received alcohol (10 mg kg^?1, water soluble) and basal diet, and group 4 received STZ and alcohol for 35 days. The cauda epididymidis of each mouse was dissected and placed in 1 ml of pre‐warm Ham's F10 culture medium for 30 min. The swim‐out spermatozoa were analysed for count, motility, morphology and viability. Sperm chromatin quality was evaluated with aniline blue, toluidine blue, acridine orange and chromomycin A3 staining. The results showed that all sperm parameters had significant differences (P < 0.05), also when sperm chromatin was assessed with cytochemical tests. There were significant differences (P < 0.001) between the groups. According to our results, alcohol and diabetes can cause abnormalities in sperm parameters and chromatin quality. In addition, alcohol consumption in diabetic mice can intensify sperm chromatin/DNA damage.     

Toxic effects of arsenic on semen and hormonal profile and their amelioration with vitamin E in Teddy goat bucks

The present environmental study has been planned to investigate the toxic effects of arsenic on reproductive functions of Teddy bucks as well as to examine whether these toxic effects are ameliorated by vitamin E. Sixteen adult Teddy bucks were divided randomly into four equal groups A, B, C and D with following treatment: A (control), B (sodium arsenite 5 mg kg^?1 BW day^?1), C (vit E 200 mg kg^?1 BW day^?1 + Arsenic 5 mg kg^?1 BW day^?1) and D (vit E 200 mg kg^?1 BW day^?1). This treatment was continued for 84 days. Semen quality parameters were evaluated weekly. Male testosterone, luteinising hormone (LH), follicle‐stimulating hormone (FSH) and cortisol levels were measured through enzyme‐linked immunosorbent assay (ELISA) after every 2 weeks. The data were subjected to two‐way analysis of variance followed by Duncan test for multiple comparisons. Semen evaluation parameters were reduced significantly (P < 0.05) in arsenic‐treated animals. The serum hormonal profile of testosterone, LH and FSH was reduced significantly (P < 0.05) in arsenic group, while the serum level of cortisol was increased. Vitamin E alleviated the toxic effects of arsenic on semen and hormonal parameters. It may be concluded from this study that sodium arsenite causes major toxicity changes in semen and hormonal profile in Teddy goat bucks and vitamin E has ameliorative effects on these toxic changes.     

Antioxidant effect of lemon balm (Melissa officinalis) and mate tea (Ilex paraguensys) on quality,lipid peroxidation and DNA oxidation of cryopreserved boar epididymal spermatozoa

In this study, we investigated the protective ability of the addition of two antioxidant herb extracts, mate tea and lemon balm, on boar epididymal frozen–thawed spermatozoa quality. Testes from mature boars were collected at local slaughterhouse, and sperm samples from epididymis were recovered by flushing. Spermatozoa were cryopreserved in lactose–egg yolk buffer supplemented with various concentrations of lemon balm and mate tea (0, 2.5, 5 and 10 g l^?1) using the straw‐freezing procedure. Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels and DNA oxidative damage (8‐hydroxy‐2′‐deoxyguanosine base lesion) were evaluated. There were no differences among experimental groups with regard to motility characteristics, viability, acrosome and plasma membrane integrity; however, the highest concentration of lemon balm produced significant (P < 0.05) improvement in curvilinear trajectory, straightness and amplitude of lateral head displacement after thawing. The supplementation of freezing extender with mate tea and lemon balm reduced sperm lipid membrane peroxidation, and only mate tea protected DNA against oxidative damage during cryopreservation at 120 min post‐thawing (P < 0.05). Mate tea experimental extender at concentration of 10 g l^?1 showed the lowest percentage of sperm oxidised DNA and malondialdehyde generation; thus, mate tea is a potential candidate such as antioxidant compound on boar sperm cryopreservation medium.     

Protective effects of udenafil citrate,piracetam and dexmedetomidine treatment on testicular torsion/detorsion‐induced ischaemia/reperfusion injury in rats

The aim of this study was to investigate the antioxidant properties of udenafil citrate (1.4 mg kg^?1–2.8 mg kg^?1), dexmedetomidine 25 μg kg^?1 and piracetam 200 mg kg^?1 administered on ipsilateral/contralateral testes after ischaemia in a rat model of testicular torsion/detorsion (T/D) and define its protective effect histologically. Fifty‐six Wistar albino rats were included and randomly assigned into 6 groups. No intervention was performed in control group (Group 1, n = 8) and in torsion/detorsion group, (Group 2, n = 8). Udenafil 1.4 mg kg^?1 was given to torsion/detorsion group (Group 3, n = 10), udenafil 2.8 mg kg^?1 was given to torsion/detorsion group (Group 4, n = 10), piracetam 200 mg kg^?1 was given to torsion/detorsion group (Group 5, n = 10) and dexmedetomidine 25 μg kg^?1 was given to torsion/detorsion group (Group 6, n = 10) intraperitoneally after 60 mins of testicular torsion. Biochemical and histopathological testicular injury were evaluated. When the tissue was examined by TOS values, Group 3, Group 4 and Group 5 were significantly lower than Group 2. In contrary Group 6 values were significantly higher than Group 2. The increasing doses of udenafil demonstrated antioxidant properties on the testis tissue and histopathological that protects the testicles.     

Melatonin prevents dexamethasone‐induced testicular oxidative stress and germ cell apoptosis in golden hamster,Mesocricetus auratus

This study investigated the protective effect of melatonin on dexamethasone (Dex), an extensively used anti‐inflammatory and immunosuppressive synthetic glucocorticoid, induced testicular oxidative stress and germ cell apoptosis in golden hamster. Hamsters were randomly divided into four groups (n = 7): group I – control; group II – melatonin treated (10 mg kg^?1 day^?1); group III – Dex treated (7 mg kg^?1 day^?1) and group IV – combination of Dex and melatonin. All the injections were administered intraperitoneally for seven consecutive days. The histopathological changes, specific biochemical markers, including antioxidative enzymes, plasma melatonin level and the markers for germ cell apoptosis were evaluated. Dex administration decreased antioxidant enzyme activities (SOD, CAT, GSH‐P_X), plasma melatonin level and melatonin receptor (MT1) expression with a concomitant increase in lipid peroxidation (TBARS) and altered testicular histopathology which might culminate into increased germ cell apoptosis as evident from increased Bax/Bcl‐2 ratio and caspase‐3 expression. However, melatonin pre‐treatment enhanced enzyme activities for SOD, CAT, GSH‐P_X with a simultaneous decrease in Bax/Bcl‐2 ratio and caspase‐3 expression. Our findings clearly suggest that melatonin improved defence against Dex‐induced testicular oxidative stress and prevented germ cell apoptosis, suggesting a novel combination therapeutic approach for management of male reproductive health.     

A comparative study of two cooling protocols on stallion sperm cryosurvival

There are many protocols for horse sperm cryopreservation, but results are inconsistent; sperm survival after freeze‐thawing is usually poor; in consequence, fertility is low. The objective of this work was to see whether slow cooling before freezing to minus 3 °C instead of +5 °C, the traditional target temperature, could improve horse sperm cryosurvival, capability to carry out capacitation and the acrosome reaction induced by progesterone. Spermatozoa from five stallions were packaged in straws and slowly cooled to +5 °C. Half of the straws were frozen directly and the other half was further cooled to ?3 °C before freezing. Progressive motility, viability, plasma membrane integrity, acrosome integrity and capacitation status were assessed. After thawing, there were no differences between cooling treatments on motility, viability, acrosome integrity and capacitation status; however, there was difference (P < 0.05) regarding plasma membrane integrity. Acrosome integrity decreased as incubation, without or with progesterone (2 μg ml^?1), progressed, but there were no differences between cooling treatments regardless of progesterone. Both capacitated and acrosome‐reacted spermatozoa increased as incubation progressed, but there were no differences between cooling treatments regardless of progesterone. Slow cooling to ?3 °C before freezing did not improve horse sperm cryosurvival or capability to undergo the acrosome reaction.     

Impact of using a fast‐freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast‐freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s^?1 or 75 °C 7 s^?1). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20 min post‐thawing in the fast‐freezing technique than in the conventional one. Plasma membrane integrity was also greater (P < 0.05) in semen frozen with the fast‐freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast‐freezing technique was 76% (19/25), and did not differ (P > 0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep‐horn insemination maximises the use of equine semen. The fast‐freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.