Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.

Semen characteristics and refrigeration in free-ranging giant anteaters (Myrmecophaga tridactyla).

The giant anteater (Myrmecophaga tridactyla) is considered vulnerable to extinction. Scientific data on the reproductive parameters of this species are scarce. Semen from eight free-ranging giant anteaters was collected to establish its characteristics and the effects of cooling and storage at 5 °C after dilution with the BotuCrio extender without cryoprotectant. The ejaculate presented two distinct sequential fractions, including a whitish fraction, which was milky and rich in sperm cells, and a gel fraction, which was colorless, viscous, and azoospermic. The mean ± standard error of the mean values of the seminal characteristics were as follows: volume of the first fraction, 0.75 ± 0.1 mL; motility, 75 ± 2.9%; vigor, 3.2 ± 0.3; sperm motility index, 68.8 ± 4.3; concentration, 108.5 ± 13.4 × 10(6)/mL; plasma membrane integrity index, 71 ± 4.0%; spermatic defects detected using modified Karras staining, 35.5 ± 3.3%; and spermatic alterations identified by differential interference contrast microscopy, 48.3 ± 6.8%. During refrigeration, the semen presented decreasing motility from 0 to 18 hours, sperm motility index decreased from 0 to 24 hours, and vigor did not change in the first 6 hours and then decreased to 18 hours.

Substituiçäo do amarelo metacromo pelo rosa bengala no método de coloraçäo para a avaliaçäo da morfologia espermática de sêmen descongelado / Substitution of the metachrome yellow by rose bengal in the staining method for the spermatic morphology evaluation of thawed semen

O presente trabalho deve como objetivo testar uma alternativa para a avaliaçäo da morfológia espermática proposta por PAPA et al. (1989), em funçäo da dificuldade de obtençäo e alto custo do corante amarelo metacromo. No Laboratório de Andrologia da F.M.V.Z. - UNESP - Botucatu foram feitos 20 esfregaços de amostras de sêmen descongelados das espécies bovina, caprina, eqüina, canina e coradas pela técnica anteriormente proposta, substituindo-se a soluçäo do amarelo metacromo glicerinada pela soluçäo de rosa bengala glicerinada (20 ml de soluçäo mäe de rosa bengala citado por MIES FILHO (1987) + 55 ml de álcool metílico + 5 ml de tampäo sorensen + 20 ml de glicerina). A análise dos esfregaços de sêmen corados com a substituiçäo proposta permitiu perfeita visualizaçäo do acrossomo, bem como das demais estruturas do espermatozóides e manteve o padräo de coloraçäo da morfologia espermática do sêmen descongelado

Alteraçöes morfológicas em ovários de éguas: tumores e hematomas / Morphological changes in the mare ovaries: tumors and hematomas

Descrevem-se as alteraçöes morfológicas diagnosticadas nos ovários de 14 éguas. Foram constatados 12 tumores das células de granulosa e 2 hematomas nos animais submetidos à ovariectomia unilateral no Hospital Veterinário da FMVZ-UNESP-BOTUCATU