Cholesterol-Loaded Cyclodextrin Addition to Skim Milk-Based Extender Enhances Donkey Semen Cooling and Fertility in Horse Mares.

The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with the combination of Yo-Pro and MitoStatusRed with flow cytometry. Semen was assessed before (0), 24 and 48h after cooling. In Experiment 2, two estrous cycles of 15 mares were used for fertility assessment. Mares were examined every other day by transrectal ultrasonography and had ovulation induced with 250 µg of histrelin acetate when a ≥35 mm follicle was first detected. Mares were randomly inseminated with semen obtained from one jack. Semen was extended in either SKM or SKM-CLC and cooled-stored for 24 hours. Pregnancy diagnosis was carried out 15-day post-ovulation. Data were analyzed with a mix model and Tukey's as posthoc and logistic regression model. Significance was set at P ≤ .05. There were no differences in TM, PM, RAP, PMS, and HMP for semen extended in either extender immediately before cooling (P > .05). There was a reduction in TM, PM, RAP, PMS, and HMP overtime across groups (P < .05); however, semen extended with SKM-CLC had superior TM, PM, RAP, PMS, and HMP than semen extended in SKM at 24- and 48-hours post-cooling (P < .05). Mares bred with semen extended in SKM had a lower conception rate (13%, 2/15 cycles) than cycles bred with SKM-CLC (47%, 7/15 cycles; P < .05). In conclusion, incorporating CLC into SKM extender improved cooling ability and fertility of donkey semen in horse mares. It remains to be determined if similar results can be obtained in clinical practice with mares and jennies.

Characterization of semen collected by pharmacologically induced ejaculation from a stallion with seminal vesiculitis.

The present study compared the quality of sperm collected by artificial vagina or pharmacologically induced ejaculation from a 10-year-old thoroughbred stallion with seminal vesiculitis. The pharmacological protocol involved intravenous administration of detomidine (0.01 mg/kg) and oxytocin (20 IU) and successfully induced ejaculation in all attempts of semen collection. Sperm motility, plasma membrane and acrosome integrity (PMAI), reactive oxygen species (ROS) levels, polymorphonuclear neutrophil (PMN) percentage, and bacterial profiles of fresh and cooled semen (5°C for 24 hr) were evaluated. Semen obtained by the pharmacological method presented reduced seminal volume, decreased PMN percentage and superior sperm motility in cooled samples. Moreover, higher PMAI and lower ROS levels were observed in semen collected by the pharmacological method. Therefore, pharmacologically induced ejaculation is an alternative to obtain semen with minimal contamination and with sperm of superior quality and longevity from stallions with seminal vesiculitis.

Sodium caseinate improves longevity and fertility of frozen bull semen.

This study aimed to evaluate the effect of sodium caseinate added into freezing extender on the sperm parameters of cryopreserved bull semen and in vitro and in vivo fertility. One ejaculate of 30 bulls was used and processed using Botu-Bov (Botupharma, Botucatu, Brazil) with the addition of 20% egg yolk (EY) or 15% egg yolk with 2% sodium caseinate (EY + SC), subsequently submitted to freezing. Semen from both groups were evaluated immediately after thawing (T0) and after thermic stress at 37 °C for 90 min (T90), for sperm kinetics, by CASA method, and plasma membrane integrity (PMI), superoxide (O2-) concentration and high mitochondrial potential (HMP) by flow cytometry. In vitro fertilization (IVF) was performed to assess embryo cleavage rate on day 3, and blastocyst rate on day 8. The in vivo fertility test was performed using fixed-time artificial insemination (FTAI). In sperm evaluation, trajectory velocity, linear velocity, curvilinear velocity, and lateral head movement were higher (P < 0.05) in EY + SC at T0. At T90, while rectilinearity and linearity did not differ between EY and EY + SC (P > 0.05), the other parameters evaluated were higher in EY + SC. Similarly, the integrity of the plasma and acrosomal membranes (iPAM) was higher (P < 0.05) at T90 in EY + SC, but did not differ (P > 0.05) between the groups at T0. For O2- and HMP, the values were lower (P < 0.05) in EY + SC group in both moments; furthermore, EY + SC showed higher cleavage and blastocyst rates in IVF. Likewise, pregnancy rates by FTAI were higher (P < 0.05) in the EY + SC group. In conclusion, the addition of sodium caseinate into freezing extender improves sperm parameters of frozen-thawed bull semen and fertility rates on during in vitro and in vivo tests.

Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions.

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

Coconut Water as an Extender Component for Cooled Equine Sperm.

The aim of this study was to evaluate the effect of coconut water as a component of extender in different formulations for cooling equine sperm. One ejaculate of fourteen stallions was collected. Sperm was diluted to 50 × 106 sperm/mL using five different extenders: ACP-105: powdered coconut water extender (ACP-105, ACP Biotecnologia, Brazil); ACP-Milk: ACP-105 + 20 g/L of skimmed milk; ACP-EY 2.5%: ACP-105 + 2.5% of egg yolk; ACP-EY 5%: ACP-105 + 5% of egg yolk; and BotuSêmen (Botupharma, Botucatu, Brazil) and cooled in passive cooling device (BotuFlex, Botupharma, Botucatu, Brazil) at 5 and 15°C for 24 hours. Sperm kinetics and plasma membrane integrity (PMI) were evaluated by computer-assisted sperm analysis and fluorescence staining, respectively, at T0 (before cooling) and T24 (24 hours after cooling). Sperm kinetics did not differ at T0 among groups (P > .05); however, at T24, these parameters were significantly lower in ACP-105 (5°C, total motility [TM]: 9.2 ± 3.6%; progressive motility [PM]: 2.7 ± 1.6%; percentage of fast-moving spermatozoa [RAP]: 4.8 ± 3.0%; 15°C, TM: 10.6 ± 3.0%; PM: 1.1 ± 0.5%; RAP: 4.8 ± 1.9%) and ACP-EY 5% (5°C, TM: 28.0 ± 6.3%; PM: 5.7 ± 1.8%; RAP: 15.9 ± 6.0%; 15°C, TM: 30.0 ± 6.0%; PM: 6.9 ± 2.1%; RAP: 17.6 ± 5.3%) compared with BotuSêmen (5°C, TM: 66.2 ± 5.6%; PM: 21.1 ± 2.8%; RAP: 53.9 ± 6.1%; 15°C, TM: 63.4 ± 5.4%; PM: 17.2 ± 2.8%; RAP: 51.4 ± 6.3%) (P < .05). All groups exhibited similar PMI at tested moments and cooling temperatures (5°C: 83%; 15°C: 84%) (P > .05). Further studies are necessary to evaluate powdered coconut water in different compositions of sperm extender; however, coconut-based extender as used in this study was not an alternative to preserve sperm parameters of cooled equine sperm.

Histrelin acetate-induced ovulation in Brazilian Northeastern jennies (Equus asinus) with different follicle diameters.

The aim of the present study was to evaluate the efficacy of a GnRH analog for induction of ovulation in Brazilian Northeastern jennies (Equus asinus) with different follicle diameters. Four consecutive estrus of 10 jennies were used in a crossover study; C (Control, n = 10) jennies were evaluated by transrectal palpation and ultrasonography until a spontaneous ovulation and the intervals between the predetermined follicular size (25-28 mm [C1], 29-32 mm [C2] and 33-36 mm [C3] follicle) and ovulation were registered. In treated cycle, jennies had the ovulation induced by 250 µg of Histrelin acetate (Strelin®, Botupharma, Botucatu, Brazil) when respective follicle diameters 25-28 mm (T1), 29-32 mm (T2) and 33-36 mm (T3) were diagnosed. Ovulation was monitored by transrectal palpation and ultrasonography. Different follicle diameters significantly affected (P < 0.05) the interval until ovulation between control and matched treated cycles. Interval between prostaglandin administration and ovulation diagnosis was lower in jennies from T2 group (145.2 ±â€¯34.6 h) compared with the control cycle (220.0 ±â€¯41.8 h) and also with other treated cycles (T1 - 209.8 ±â€¯48.0 h; T3 - 183.3 ±â€¯33.9 h). Histrelin acetate treatment also reduces the interval between detection of predetermined follicular size and ovulation (P < 0.05) in all treated cycles groups compared with matched control group. Higher percentage (P < 0.05) of jennies had success of ovulation induction (36-48 h after Histrelin acetate injection) in all treated cycles in contrast with the matched control group. In addition, in comparison among treated cycle groups, more (P < 0.05) jennies (100%) in T2 ovulated between 36 and 48 h after ovulation induction, compared with T1 and T3, which did not differ (P > 0.05) from each other. Edema scoring and ovulation were not associated events (r = 0.0219). In conclusion, jennies with 29-32 mm follicles satisfactory responded to ovulation induction with Histrelin acetate, which allowed the shortening of interovulatory interval in all groups evaluated.

Can centrifugation force compromise the plasmatic membrane, acrosome and DNA integrity of goat spermatozoa? / A força de centrifugação pode comprometer a integridade de membrana plasmática, acrossomal e DNA de espermatozoides caprinos?

Protocols for cooling or freezing goat semen usually recommend centrifugation for seminal plasma removal. However, little is known about the effect of this process on goat sperm viability and functionality. The present study evaluated the effects of centrifugation force on the plasma membrane, acrosomes, and DNA integrity of goat semen. Four ejaculates from each of the four different Anglo Nubian male goats were used. Semen samples were obtained using artificial vagina, and immediately after collection, ejaculates were diluted using Ringer's sodium lactate solution and split into three groups: Control (CG, without centrifugation), G1 (centrifugation 600 x g/10 min), G2 (centrifugation 1200 x g/10 min). After centrifugation, seminal plasma was removed, the sperm pellets were resuspended using Tris-egg yolk extender (80 x 106 spermatozoa/mL) and the sperm morphology was analyzed. Samples were cooled at 5°C for 5, 24, 36, and 48 h and then sperm plasma membrane and acrosome integrity (PMAI, %) and sperm DNA fragmentation index (SDF, %) were evaluated at each time-point, using a flow cytometer. Additionally, sperm movement was determined using computer semen analysis (CASA) after 5, 24, and 48 h of refrigeration period. The semen centrifugation did not induce additional sperm morphology defect or reduction in sperm kinetics in the experimental groups. Differences were not observed (p > 0.05) in PMAI and SDF among different groups, in any of each time-point of the cooling process. In conclusion, centrifugation, even at high speeds, did not affect goat sperm integrity and functionality when submitted to refrigeration process. (AU)

A maior parte dos protocolos de refrigeração e criopreservação do sêmen caprino recomenda o uso de centrifugação para remoção do plasma seminal. No entanto, não existe consenso sobre o risco que esse tipo de processamento pode ocasionar à viabilidade espermática. Nesse contexto, o presente trabalho investigou os possíveis efeitos deletérios da centrifugação sobre a integridade estrutural e DNA de espermatozoides caprinos. Para a pesquisa foram selecionados quatro reprodutores para colheita de sêmen (n = 4 ejaculados/bode). Cada ejaculado foi fracionado em três alíquotas iguais, diluídas em ringer e divididas em três grupos: Controle (GC, não centrifugado), G1 (centrifugação a 600 g/10 minutos) e G2 (centrifugação a 1200 g/10 minutos). As amostras seminais por grupo foram diluídas em meio Tris gema respeitando-se a concentração final de 80 milhões de espermatozoides/mL e foram submetidas à avaliação de morfologia espermática. Todas as amostras foram acondicionadas a 5°C, sendo analisadas nos momentos 5, 24, 36 e 48 horas do processo de refrigeração por meio da avaliação da integridade de membrana plasmática e acrossomal (MPAI, %) e índice de fragmentação de DNA (IDF, %). Adicionalmente, a cinética espermática foi avaliada com o emprego de um sistema computadorizado de análise (CASA) nos momentos 5, 24 e 48 horas da refrigeração. A centrifugação não induziu a manifestação de defeitos morfológicos ou redução significativa da cinética de espermatozoides caprinos. Não foram observadas diferenças para a integridade de membrana plasmática e para o índice de fragmentação de DNA quando comparados, respectivamente, GC, G1 e G2 em cada um dos quatro momentos experimentais. Conclui-se que mesmo quando empregadas altas forças de rotação não ocorre lesão à ultraestrutura dos espermatozoides caprinos submetidos ao processo de refrigeração.(AU)

Morphometric changes in the hoof capsule of Criollo foals from birth to weaning / Alterações morfológicas na capsula do casco em potros Crioulos do nascimento ao desmame

ABSTRACT: South America has numerous Criollo horse breeding farms; however, information on foal hoof growth is still limited and identifying the ideal periods to apply corrective trimming is a frequent concern for horse owners. In the present study, a morphometric analysis of hoof growth was performed on 46 Criollo foals from birth to weaning (0-8 months). Results showed that hoof growth rate was higher in the first four months followed by a decrease until the eighth month. Average growth rate of the hoof was 0.21cm per month, whereas that of the heel was 0.14cm per month. However, no significant differences were observed between medial and lateral heel length or between limbs. Coronary band perimeter and solar width showed an average increase of 0.97cm and 0.46cm per month, respectively, and were significantly correlated (r=0.99, P≤0.01). The characteristic most positively correlated to biometric variables was foal age, followed by solar width, toe length, and coronary band perimeter. In conclusion, hoof length increase in Criollo foals was more intensive during the first four months of life.

RESUMO: A América do Sul possui um grande número de criatórios da raça Crioula, no entanto, há uma carência de informações sobre o desenvolvimento natural dos cascos dos potros, tornando a identificação de períodos ideais para o casqueamento corretivo uma dúvida frequente entre os criadores. Portanto, o objetivo deste estudo foi realizar a biometria natural (do nascimento aos 8 meses) no casco de 46 potros da raça Crioula. Os resultados indicaram uma taxa de crescimento mais rápido nos primeiros 4 meses, com subsequente desaceleração até o desmame. O crescimento médio do casco foi em média 0,21cm mo-1, enquanto o comprimento do talão foi de 0,14cm mo-1. Não foram observadas diferenças significativas no equilíbrio médio/lateral do casco ou entre os membros durante o período experimental. O perímetro da banda coronária e a largura solar do casco apresentaram um crescimento médio de 0,97 e 0,46cm mo-1, respectivamente, e foram altamente correlacionados (r=0,99, P≤0,01). A idade dos potros foi a característica mais correlacionada positivamente, seguida da largura solar, do comprimento do casco e do perímetro da banda coronária. Nós concluímos que o crescimento do casco em potros da raça Crioula foi mais intenso durante os quatro primeiros meses de vida.