Effects of insulin on sperm cell quality in ram semen cooled at 5°C

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (μm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.

Recovery of Prenatal Baicalein Exposure Perturbed Reproduction by Postnatal Exposure of Testosterone in Male Mice

Baicalein (BC), a flavonoid, which lacks the qualities of reproductive health and shows adverse effects, is tested in this study. Inseminated mice were injected with 30, 60, and 90 mg BC/Kg body weight on gestation days 11, 13, 15, and 17. The F1 BC-exposed males of each dosage were divided into six groups. First three groups (n = 6 from each BC dosage) were used for assessment of reproductive performance, the others (n = 4 from each BC dosage) were administered with testosterone 4.16 mg/kg body weight on postnatal days 21, 31, and 41. The reproductive health of adult F1 males at the age of 55 and 60 was tested. Prenatal BC exposure showed reduced fertility after cohabitation with control females. The BC exposure significantly reduced the body weight, tissue indices, and sperm parameters (motility, count, viability, and daily sperm count) and altered the sperm membrane in a hypoosmotic swelling test. A downward trend was observed in testicular steroidogenic marker enzymes (3β- and 17β-steroid dehydrogenases) and serum testosterone, whereas increase in serum titers of FSH and LH along with altered the testicular histology. Conversely, testosterone (4.16 mg/kg body weight) partially recovered reduced male reproductive health by BC. BC impaired male reproductive health due to low levels of testosterone is reverted by external testosterone is evidenced in this study.

Progesterone decrease plasma membrane in human sperm with subnormal hypoosmotic swelling test scores

Background<br />Progesterone (P4) is known as a female hormone affecting oocyte maturation and developing uterine wall. A proteomic study identified several receptors including P4 receptors on human sperm. The role of P4 in human sperm cells remains unknown as to whether P4 has non-genomic effects on human sperm. The present study aims to determine the effect of progesterone (P4) on the hyperactivated motility and membrane integrity of human sperm cells.<br /><br />Methods<br />Semen from normal individuals was obtained from donors. The semen was washed by gradient density centrifugation. P4 was added to each semen sample to final concentrations of 0 (control), 250, 500, 750 and 1000 ng/mL. After the sample treatment was completed, the sperm membrane integrity was assessed with the hypoosmotic swelling test (sodium citrate dihydrate and D-fructose) and the hyperactivated sperm motility parameter was determined with the Computer Assisted Sperm Analyzer [CASA] (Hamilton Thorne, IVOS II, USA). The percentage was then compared between the treatment groups and the control group. The percentage differences were analyzed with the Sigmastat version 2.0 statistical program.<br /><br />Results<br />Administration of P4 increased sperm hyperactivated motility when compared with the control group at a concentration of 500 ng/mL, but the increase was statistically not signicant (p&gt;0.05). In contrast, P4 decreased sperm membrane integrity significantly (p=0.042). And the mean of plasma membrane integrity in all groups was subnormal hypoosmotic swelling test score. <br /><br />Conclusion<br />Progesterone administration tends to increase sperm hyperactivated motility. The integrity of plasma sperm membrane was affected by progesterone.

Functional sperm assessments of African Lion Panthera leo (Mammalia: Carnivora: Felidae) in field conditions

Wild African Lion Panthera leo populations are decreasing due to inbreeding and reduced genetic variability. Thus, the use of assisted reproduction in the species could one day become essential. Before this is possible, however, studies need to be conducted on the basic reproductive traits of the species, especially those regarding sperm cells. This study aimed to analyze the semen of African Lions in field conditions. We included seven captive African Lions in our study. The animals were chemically restrained and electro-ejaculated. Twenty sperm samples were selected and analyzed for sperm motility and progressive motility, sperm motility index, and sperm morphology. In addition, the samples were analyzed for membrane and acrosome integrity (hypoosmotic swelling test and fast green/rose Bengal dyes, respectively) and assessed for cytochemical activity of the mitochondria. We found that sperm motility rate was 75.25%±2.03, progressive motility rate was 3.25%±0.10, and sperm motility index was 70.12%±1.71. We found morphologic abnormalities roughly at the expected rate with 34.61%±7.22 of the sperm cells having an intact plasma membrane and acrosome integrity of 92.27%±2.73; high mitochondrial activity was 54.26±4.88% and absence of mitochondrial activity was 2.72±0.68% in the sperm cells. These findings show that conventional tests for sperm motility and sperm morphology bring about the expected results for lions according scientific literature. Though a hypoosmotic swelling test may be performed using different concentrations, it might lead to a higher number of sperm cells with membrane damage. Fast green/rose Bengal stain and 3’3 diaminobenzidine assay, however, can be used in sperm analysis of lions in field conditions.

Effect of thawing method on bull sperm survival in ejaculates frozen in 4 ml and 8 ml volumes

The aim of this study was to evaluate the effect of different thawing protocols (slow (P1), medium (P2), and fast (P3)) on percentage of motile sperm (MOT) and percentage of sperm cells with intact membranes (INT) in Holstein (4 bulls; 72 samples) and Czech Fleckvieh (4 bulls; 72 samples) semen frozen-thawed in 4 ml and 8 ml volumes. MOT was analysed in fresh semen, as well as immediately after thawing (T0) and 30 min after thawing (T30). INT was analysed using hypoosmotic swelling test (HOS test) in fresh ejaculate (FE), after diluting (DE), and at T0. The differences between FE parameters and frozen-thawed ejaculate parameters, expressing changes that occur during cryopreservation, were calculated. Apart from the effect of thawing protocol, the effect of breed and the effect of quality of FE expressed by MOT immediately after collecting were evaluated, too. Unlike thawing of semen cryopreserved in straws (0.25 and 0.5 ml), thawing using the slow protocol (P1) was the most appropriate (P &lt; 0.05) for both observed volumes. There were found significantly higher MOT in the volume of 8 ml in both T0 and T30 and in the volume of 4 ml in T30 in samples thawed using P1 and P2. MOT in T0 was significantly affected by breed in samples frozen in 8 ml and in T30 in samples frozen in 4 and 8 ml. There were found no significant differences in INT in all reported volumes, however decrease of INT during cryopreservation was affected by breed.

Effect of different extenders for preservation of ram semen at 4°C

The aim of this study was to investigate the effects of different extenders viz. Tris citric acid fructose egg yolk (TCFEY), Tris citric acid glucose egg yolk (TCGEY), Egg yolk citrate fructose (EYCF) and Egg yolk citrate glucose (EYCG) on the quality of ram spermatozoa during preservation at 4°C. Semen samples showing more than 3+ mass motility and 70% progressive motility were pooled and subsequently divided into four aliquots. Each aliquot was extended separately in four different extenders viz. TCFEY, TCGEY, EYCF and EYCG and stored at 4°C up to 72h. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, morphological abnormalities, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated immediately after extension in particular extenders (0 h), 24 h, 48 h and 72 h after preservation at 4°C. The percent sperm motility was significantly (P<0.01) higher for TCFEY and TCGEY than EYCF and EYCG at 72 h of preservation at 4°C. The percent HOST reacted spermatozoa and intact acrosomes were significantly (P<0.01) higher and morphological abnormalities were significantly (P<0.01) lower for Tris based fructose extender than other three extenders at 72 h at 4°C. In conclusion, Tris citric acid fructose egg yolk (TCFEY) was found the best in maintaining the quality of ejaculated ram spermatozoa during preservation for 72 h at 4°C.