S. V. Silva

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.

Efeito da temperatura de descongelação na integridade de espermatozoides criopreservados de cães

Aiming to evaluate the influence of the thawing temperature on the viability of canine cryopreserved sperm, Basset Hound (n=3) and Rottweiler (n=3) dogs were used, submitted to semen collected through manual manipulation. Semen samples were thawed at 37oC during 1min (G1) or at 70oC during 6s (G2), and evaluated for progressive motility, vigor and acrosome integrity, after 0, 30 e 60 minutes of incubation (37oC), and sperm ultrastructure immediately after thawing. In all incubation times, the average of progressive motility was higher (P 0.05) between groups, and the percentage of gametes with intact acrosome was higher (P<0.05) on sperm cells from G1 than from G2. Ultrastructural changes were identified on dog sperm from both groups, and were observed in higher quantity in gametes from G2 Group. It can be concluded that samples of frozen dog sperm must be thawed at 37°C for 1min.

Effects of glycerol, equilibration time and antioxidants on post-thaw functional integrity of bovine spermatozoa directly obtained from epididymis

This work aimed to evaluate the effect of stabilisation times, glycerol concentration, and the catalase and superoxide dismutase supplementation of diluent on parameters of frozen‐thawed spermatozoa from epididymis of Nelore bulls: Experiment 1: spermatozoa diluted in Tris‐egg yolk with glycerol (3%, 5% or 7%) and stabilisation times (0, 2 or 4 hr at 5°C); Experiment 2: Tris‐egg yolk only, Tris‐egg yolk with catalase (CAT, 50 or 100 U ml−1) or superoxide dismutase (SOD, 50 or 100 U ml−1). Frozen‐thawed spermatozoa were evaluated for kinetic parameters, plasma membrane and acrosome integrity, mitochondrial activity and IVF capacity. ALH and BCF were affected (p < .05) by glycerol at 3% after 4‐hr equilibration time and 7% after 2‐hr equilibration time. Glycerol 3% had lower (p < .05) iPM and iAc after 4 hr. Glycerol 5% had greater (p < .05) hPMM after 4 hr and iAc after 2 hr than at 0 hr. SOD 100 U ml−1 had lower (p < .05) linearity and wobble compared to control group. No was observed differences to fertilisation rate (p < .05) among groups. In conclusion, glycerol 5% in Tris‐egg yolk extender for 4 hr is suitable for the preservation of sperm kinetics and membrane integrity. CAT (50 and 100 U ml−1) or SOD (50–100 U ml−1) had no beneficial effects on sperm kinetics, plasma and acrosomal membrane integrity, mitochondrial activity or the capacity for IVF of frozen‐thawed spermatozoa from epididymis of Nelore bulls.

High resveratrol or quercetin concentrations reduce the oscillation index of frozen goat semen

O objetivo deste estudo foi avaliar o efeito de diferentes concentracoes de transresveratrol ou quercetina sobre a capacidade dos espermatozoides caprinos de resistirem a congelacao. Seis pools de semen, obtidos de seis reprodutores caprinos, foram processados com diferentes concentracoes de resveratrol ou quercetina (Experimento 1: 0, 15, 25, 50, 75 ou 100μM de resveratrol; Experimento 2: 0, 15, 25, 50, 75 ou 100μM de quercetina) e congelados. Apos o descongelamento, o semen foi avaliado quanto a cinetica espermatica, a integridade das membranas plasmatica e acrossomal, a morfologia e ao estresse oxidativo nos tempos zero e uma hora de incubacao. Imediatamente apos a descongelacao (zero hora), o wobble (indice de oscilacao) nos grupos tratados com 100μM de quercetina ou de resveratrol foi menor (P<0,05) do que nos tratados com 0 e 25μM de resveratrol e com 0μM de quercetina, respectivamente. Apos uma hora de incubacao, a motilidade total dos tratamentos com 15, 50 e 75µM de quercetina, assim como a integridade de membrana plasmatica em todas as concentracoes de quercetina, foi menor (P<0,05) do que a zero hora. Em oposicao, a linearidade das amostras de semen tratadas com 100µM de quercetina e a retilinearidade daquelas tratadas com 75µM e 100µM de quercetina foram menores (P<0,05) a zero hora do que a uma hora apos descongelacao. Assim, pode-se concluir que o resveratrol e a quercetina, em concentracoes elevadas (100μM), reduzem, transitoriamente, o indice de oscilacao de espermatozoides caprinos submetidos a congelacao e que a quercetina (75 e 100µM) aumenta a linearidade e a retilinearidade ao longo do tempo, o que pode ser favoravel a fertilidade.

Activity of crude extract of the sexual renal segment from Crotalus durissus on spermatic kinematics of thawed dog semen

Background Males of the order Squamata have similar structure to the seminal vesicles of mammals called Renal Sexual Segment (RSS). The RSS regions are hypertrophied kidney ducts, androgen-dependent, responsible for providing secretions (complex of glycogen, mucopolysaccharides, mucoproteins and lipids) that nourishes and keeps the sperm during copulation [1,3], acting accessorily on reproductive cycle, once when mixed with sperm, and nutrient function, also act as activating agent of sperm transferred to the cloaca in the female during intercourse [2,3]. Valverde (personal communication, April 6, 2010) tested the crude extract of the renal sexual segment Crotalus durissus in bovine semen and the results showed a significant increase in sperm motility and vigor of the samples, but this activity was lost over time storage of the extract. The promising data in this study aimed to evaluate the effect of the addition of crude extract of the RSS from C. durissus on spermatic kinematics of thawed dog semen.

Cryopreservation of ram epididymis spermatozoa post-slaughter - A feasible biotechnique?

Background Gametes for breeding high-value livestock is commonly preserved in gene banks for later use in assisted reproduction programs. Such material can be used in artificial insemination or embryos transfer in the field as well as embryos in vitro production [1]. However, when these animals die, genetic material may be lost if it is not possible to recover and preserve spermatozoa from epididymis. Thus, our aim is to establish a recovery technique of ram sperm epididymal post-slaughter and to test its postcryopreservation viability.