Felipe Costa Almeida

Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen

The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.

Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation

Several studies reveal that vitamin E acts as a cellular stabilizer of unsaturated lipids against oxidative deterioration, thus maintaining structural and functional integrity at the subcellular level. The objective of this study was to evaluate Vitamin E (Trolox) addition to freezing extender for ram spermatozoa. Semen samples were diluted in Tris-yolk egg medium without antioxidant (control group) and with Trolox in different concentrations (30, 60 and 120μM). After thawing (37°C/30s), samples were subjected to analysis for plasma membrane integrity (PMi), acrosome integrity (Aci), mitochondrial membrane potential (MMP), sperm kinematics, and ultrastructural integrity. The Trolox 60 and 120μM groups showed higher percentages of iPMs (P<0.05) when compared to the control group. Differences were observed among groups in sperm kinematic indicators (progressive motility, linearity, straightness, oscillation index, straight-line velocity and average path velocity), with higher values (P<0.05) for the Trolox 60 and 120μM groups. On ultrastructural assessment, Trolox addition at the three concentrations preserved spermatozoon head plasma membranes, while for the spermatozoon tail, plasma membrane preservation at 60μM was higher (P<0.05) than the other groups. The Trolox 60 and 120μM groups presented more mitochondrial ultrastructural preservation than the other groups (P<0.05). These results indicate that Trolox addition to Tris-egg yolk at 60 and 120μM provides greater structural integrity (plasma membrane and mitochondria) and kinematics for ram spermatozoa after cryopreservation.

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 adição de glutationa peroxidase e cisteína ao diluidor de congelação do sêmen equino

Ejaculates (n=25) of horses were used to assess the effect of glutathione peroxidase (GPx) and cysteine on the viability of frozen sperm cells. Semen was extended at Botu Crio with antioxidants, and divided in groups: G1, control; G2, 1 U GPx; G3, 5U GPx; G4, 0.5mM cysteine and G5, 1mM cysteine, packed in 0.5mL straws, and frozen. After thawing (37° C for 30 seconds) samples were analyzed for plasma membrane (IMP) and acrosome integrity (IAc), mitochondrial membrane potential (MMP) and kinematic, at zero (T0) and 60 minutes after (T60). GPx 5U and cysteine 0.5mM increased (P<0.05) IAc at T0, when compared to T60. Cysteine 1mM resulted in a higher (P<0.05) IAc on T60, than GPx 1 and 5U, and cysteine 0.5mM. The PMM from a stallion on T60 was higher (P<0.05) than those of two stallions. In sperm kinematic, VCL and VAP were higher (P<0.05) at T0 compared to T60 for the control group, and one stallion showed larger (P<0.05) kinematic values than other animals. It is concluded that the addition of glutathione peroxidase at concentrations 1U and 5U, and cysteine, at concentrations of 0.5mM and 1mM, does not interfere with the integrity of cryopreserved equine sperm, but preserves the kinetic parameters VCL and VAP after 60 minutes of incubation. It should be noted also that the stallion has a strong influence on sperm characteristics post-freezing.

Espermatozoides caprinos criopreservados em meio à base de leite desnatado acrescido de glutationa reduzida

Aiming to evaluate in vitro effect of different concentrations of glutathione reduced (GSH) in skimmed-milk and glycerol 7% it was used semen from five Boer bucks. After collect and evaluation, a pool of samples was diluted in skimmed-milk and glycerol 7% plus antioxidant: G1) Control; G2) GSH 2mM mL-1; G3) GSH 5mM mL-1 and G4) GSH 7mM mL-1. Samples were frozen in straws (0.25mL) and stored at -196°C. After thawing, samples were subjected to integrity of the plasma membrane (iMP) and acrosomal (iAc), mitochondrial membrane potential (MMP), kinematic and ultrastructure analysis. Control and GSH (2, 5 and 7mM mL-1) groups did no differ (P>0.05) in iMP, iAc, PMM and kinematic parameters. In the ultrastructural analysis, percentages of acrosome and plasma membrane (tail and head region) intact did not differ (P>0.05) between groups. However, Control group had higher percentage (P<0.05) of gametes with intact axonemes than those of GSH (2, 5 and 7mM mL-1) groups. Higher percentage (P<0.05) of sperms with intact mitochondrias were observed on Control group than those of GSH (5 and 7mM mL-1). It can be concluded that the GSH (2, 5 and 7mM mL-1) addition in skimmed-milk diluent to freeze goat semen did not preserve sperm integrity.

Interferência da condição climática na integridade de espermatozoides ovinos submetidos à criopreservação

Avaliou-se a integridade de espermatozoides ovinos colhidos e criopreservados em diferentes situacoes climaticas na regiao semiarida do estado da Paraiba. As colheitas de semen foram realizadas em duas epocas do ano, periodo seco e periodo chuvoso, utilizando cinco machos adultos da raca Santa Ines, com historico favoravel de fertilidade. Os ejaculados foram avaliados quanto a motilidade, vigor, concentracao e morfologia espermatica, apos a formacao do pool e diluicao em Tris-gema, na concentracao final de 240x106 espermatozoides/mL. Motilidade total e vigor espermatico nao diferiram entre as epocas seca e chuvosa. Valores de integridade do acrossoma e da membrana plasmatica, e o potencial de membrana mitocondrial foram mais baixos (P<0,05) na epoca com menor indice pluviometrico. Conclui-se que a reproducao de ovinos criados na regiao do semiarido paraibano sofre acao da condicao climatica e sugere-se que a criopreservacao de espermatozoides ovinos seja realizada no periodo de maior indice pluviometrico na regiao.

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.

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.