V. H. Barnabe

Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm2 of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r2 = 0.91 and CASA: r2 = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Semen evaluation of Murrah buffalo bulls using sperm functional tests

Abstract The aim of this experiment was to evaluate membrane integrity, vitality, and mitochondrial cytochemical activity, in frozen semen samples of buffalo bulls and compare those functions with the routine semen evaluation and field fertility. Twenty one frozen semen batches from 2 buffalo bulls were used for AI. For the semen evaluation, after thawing, an aliquot was evaluated for motility and vigor. An aliquot of each batch was used to evaluate the cytochemical activity using the 3-3’ diamino benzidine. Samples were scored in four classes according to the degree of midpiece staining, being class I showing midpiece totally stained, indicating full mitochondrial activity, and class IV showing no staining of the midpiece, indicating no mitochondrial activity. Two other aliquots were used for the hypo-osmotic swelling test (HOST) and the eosin nigrosin staining (VIT), to evaluate membrane integrity and vitality, respectively. Correlations were found between pregnancy rate and vitality and class II and III of the DAB staining (r=0.53, r=-0.39, and r=-0.38, respectively; p=0.05). No correlation was found for pregnancy rate and motility or vigor. Results indicate that functional tests may be an alternative to better predict the fertility of buffalo frozen semen samples.

Extender Supplementation with Antioxidants Selected after the Evaluation of Sperm Susceptibility to Oxidative Challenges in Goats

ABSTRACT This study aimed to detect the most deleterious ROS for goat sperm and then supplemented the extender with a proper antioxidant. For this, 12 adult goats (aged 1–7) were used. Fresh samples were submitted to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and malondialdehyde (MDA—toxic product of lipid peroxidation). After experiment 1, sperms were cryopreserved in extenders supplemented to glutathione peroxidase (Control: 0 UI/mL; GPx1: 1 UI/mL; GPx5: 5 UI/mL, and GPx10: 10 UI/mL) and catalase (Control: 0 UI/mL; CAT60: 60 UI/mL; CAT120: 120 UI/mL, and CAT240: 240 UI/mL). Each sample was evaluated by motility, plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, assay of the sperm chromatin structure, mitochondrial activity (3,3-diaminobenzidine), and measurement of lipid peroxidation (thiobarbituric acid reactive substances [TBARS]). It was possible to observe a mitochondrial dysfunction (DAB—Class IV) and low membrane integrity after hydrogen peroxide action. However, the high rates of TBARS were observed on hydroxyl radical. CAT240 presents the lower percentage of plasma membrane integrity. It was possible to attest that hydrogen peroxide and hydroxyl radical are the more harmful for goat sperm. Antioxidant therapy must be improving perhaps using combination between antioxidants.