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Made available in DSpace on 2022-04-28T17:22:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-09 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Seminal plasma has several components that protect the sperm cells and assist in the fertilization process. In contrast, the exact role carried out by seminal plasma during the cooling of canine semen remains controversial. Moreover, concerning the long estrus period, the possibility to store chilled semen at 5 degrees C for more than 72 hours and maintain good sperm quality for additional inseminations could increase fertilization rates. Thus, this study aimed to evaluate the seminal plasma influence on quality and oxidative stress of the extended canine semen stored at 5 degrees C for 7 days. Three ejaculate pools from eight healthy dogs were collected by digital manipulation of the penis. The sperm kinetics, sperm vitality (eosin/nigrosin stain), integrity of plasma and acrosomal membranes, morphology, superoxide and hydrogen peroxide production, mitochondrial potential, lipid peroxidation, and oxygen reactive species production (induced and spontaneous thiobarbituric acid [thiobarbituric acid reactive substances, TBARS] assay) were evaluated every 48 hours (M0, M48, M96, and M168) until 7 days (168 hours) in cooled extended (TRIS egg yolk) semen of dogs at 5 degrees C with (+SP) or without (-SP) autologous seminal plasma. No statistical difference was found for sperm kinetics in cooled samples with +SP and -SP during the experimental time period, except for the progressive motility of +SP samples that was higher at M48 than M96 (p = 0.023). The seminal plasma did not influence any other evaluated sperm characteristics. Finally, our results demonstrated that the presence or lack of seminal plasma during cooling the semen of dogs does not influence sperm quality at 5 degrees C. Moreover, the components of the semen extender may contribute to maintaining good sperm quality and low reactive oxygen species production during the long period of the dog's semen cooling, even after semen centrifugation. Sao Paulo State Univ UNESP, Fac Vet Med & Anim Sci, Dept Vet Surg & Anim Reprod, Rua Prof Dr Walter Mauricio Correa S-N, BR-18681681 Botucatu, SP, Brazil Univ Brasilia, Inst Biol Sci, Dept Physiol Sci, Brasilia, DF, Brazil Univ Sao Paulo, Fac Vet Med & Anim Sci, Dept Anim Reprod, Sao Paulo, Brazil Sao Paulo State Univ UNESP, Biosci Inst, Dept Biostat, Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Fac Vet Med & Anim Sci, Dept Vet Surg & Anim Reprod, Rua Prof Dr Walter Mauricio Correa S-N, BR-18681681 Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Biosci Inst, Dept Biostat, Botucatu, SP, Brazil FAPESP: 2017/02530-8 CAPES: 33004064086P1

Made available in DSpace on 2022-04-29T08:32:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio® (Botupharma, Botucatu, Brazil) or Tris–yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris–yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) University of Franca (UNIFRAN) Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP)

Os garanhões possuem um conjunto completo de glândulas sexuais acessórias, compostas pelas bulbouretrais, a próstata, vesículas seminais e ampolas dos ductos deferentes. Dentre as afecções que acometem essas estruturas, a vesiculite seminal é a de maior ocorrência, consistindo na colonização de uma ou ambas as vesículas por bactérias, sendo a Pseudomonas aeruginosa a mais frequente. No ejaculado é observada uma grande percentagem de neutrófilos e, eventualmente, hemácias podem estar presentes, caracterizando os quadros de piospermia e hemospermia, respectivamente. O diagnóstico definitivo é realizado por meio da endoscopia transuretral das vesículas, onde é possível visualizar o conteúdo purulento, associado à cultura bacteriana do lavado da glândula. O tratamento é desafiador, pois a antibioticoterapia apresenta baixa eficácia, com altas taxas de recidiva.

Made available in DSpace on 2021-06-25T11:07:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. School of Veterinary Medicine and Animal Science Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science Sao Paulo State University (UNESP) FAPESP: 2016/21452-5

Made available in DSpace on 2020-12-12T02:47:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Treatments for seminal vesiculitis have poor outcomes in stallions; thus, the development of alternative strategies is warranted. This study aimed to evaluate fractionated semen collection as a method to restore the fertility of stallions diagnosed with seminal vesiculitis. Eighteen ejaculates from six stallions (three ejaculates/stallion) diagnosed with seminal vesiculitis were harvested in fractions, as follows: Fraction A (FA), the first two jets; Fraction B (FB), the third and fourth jets; and Fraction C (FC), the fifth and remaining jets of the ejaculate. All fractions were subject to standard semen evaluations that were performed in addition to cytology and bacterial aerobic cultures. Fractions were extended and cooled to 5 °C. As a proof of concept, 20 mares (48 estrous cycles, ∼8 cycles/stallion) were bred with 1 billion sperm from FA (cooled at 5 °C for 24 h). In our study, FA had negative bacterial cultures, absent macroscopic or microscopic abnormalities; FB had positive bacterial cultures in two stallions and presence of polymorphonuclear neutrophils (PMNs) in all samples, but with no macroscopic abnormalities; and FC had positive bacterial cultures, purulent appearance, and the presence of degenerated PMNs, just as noted in the whole semen. Overall, post-cooling sperm motility results were superior (P < 0.05) for FA in comparison with FB and FC. First cycle pregnancy rates using FA varied from 66% to 86%. None of the non-pregnant mares developed endometritis. In conclusion, fractionated semen collection can be used to obtain semen free of contamination and to achieve satisfactory pregnancy rates from stallions with seminal vesiculitis. São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science FAPESP: # 2014/00354-0

Made available in DSpace on 2020-12-12T02:47:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control—pure raw semen, (b) WB50—50% (v/v) whole blood added into semen, (c) E1—WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2—WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O2) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14 days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p .05); however, O2 production was higher (p .05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p

The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 10

Made available in DSpace on 2022-05-01T13:41:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-04-15 This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. Significance: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species. São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science

The development of techniques to increase sperm longevity demands knowledge about cellmetabolism. Sperm requires a constant supply of energy to maintain its cell functions.Approximately 500 metabolic reactions take place in somatic cells, and several of them requireenergy. Most of the produced energy goes for sperm motility, which is an ATP-dependentspecialized process. Spermatozoa possess the required mechanisms to produce energy throughglycolysis, citric acid cycle (Krebs cycle) and oxidative phosphorylation. Understanding thesepathways provides knowledge on the interactions between the semen extender substrates andsperm cells. The aim of this review is to approach the major energy producing pathways of thesperm, as well as the substrates available for this metabolism.

Made available in DSpace on 2019-10-06T17:13:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-15 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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 ± 34.6 h) compared with the control cycle (220.0 ± 41.8 h) and also with other treated cycles (T1 - 209.8 ± 48.0 h; T3 – 183.3 ± 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. Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science Federal University of Bahia (UFBA) Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science

Made available in DSpace on 2019-10-04T12:35:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Boar semen cannot be immediately cryopreserved, it need be hold at 17 degrees C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 degrees C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 degrees C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h. Univ Sao Paulo, Swine Res Ctr, Sch Vet Med & Anim Sci, Pirassununga, SP, Brazil Sao Paulo State Univ, Sch Vet Med & Anim Sci, Dept Anim Reprod & Vet Radiol, Botucatu, SP, Brazil Sao Paulo State Univ, Sch Vet Med & Anim Sci, Dept Anim Reprod & Vet Radiol, Botucatu, SP, Brazil FAPESP: 2015/17620-7 FAPESP: 2016/09441-8 FAPESP: 2016/24690-4

Made available in DSpace on 2019-10-06T16:05:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Studies involving different methods and techniques of cryopreservation and its interactions with the conception rates in artificial insemination (AI) programs are reported in the literature. This study evaluated the sperm kinetics, plasma membrane integrity, and fertility rates of mares inseminated with cryopreserved stallion semen subjected to different freezing methods. For this, four ejaculates from five stallions were collected and frozen in conventional (Styrofoam box) or automated system in Mini-Digitcool ZH 400. Seminal samples were evaluated after thawing for sperm motion parameters by CASA and plasma membrane integrity by epifluorescence microscopy. For the fertility trial, a cross-over model was performed using 100 cycles of 50 mares, which were inseminated by one the two freezing methods. No differences were observed for sperm motion parameters and plasma membrane integrity between groups (P >.05). The pregnancy rate using the conventional method was 56% (28/50) and did not differ (P =.5406) from the pregnancy rate (64%, 32/50) obtained using the automatized method. The use of semen from fertile stallions may not illustrate small differences in the two freezing methods evaluated. Conventional and automated freezing systems did not differ in the quality and viability of fertile stallion semen and conception rates, indicating that the two methodologies can be safely used in AI programs. Department of Animal Reproduction Parananense University Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP Department of Animal Reproduction Minas Gerais Federal University Department of Animal Science Uberaba University Department of Animal Science Santo Amaro University Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP

Made available in DSpace on 2022-04-29T08:35:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study aimed to evaluate the cryopreservation effects on the semen of oncilla (Leopardus guttulus, n = 5, 15 ejaculates) and ocelot (Leopardus pardalis, n = 5, 17 ejaculates) and compare two extenders (commercial and non-commercial extender). An andrological exam was conducted (testicle measurements and penis evaluation), including semen collection by electroejaculation. After collection, the semen was assessed to volume, color, pH, sperm motility, vigor, sperm number in the ejaculate, viability, membrane integrity, and sperm morphology. Samples were centrifuged (300 g for 10 min) and pellet diluted in two extenders (TRIS/glucose/egg yolk and BotuCRIO®), packed into 0.25 mL French straws (20 × 106 spermatozoa/mL), equilibrated at 5 °C for 1 h (

Made available in DSpace on 2020-12-12T02:45:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 The goal of this study was to compare the efficiency of histrelin acetate (GnRH analog) and human chorionic gonadotropin (hCG) to hasten ovulation in Brazilian Northeastern jennies (Equus africanus asinus). Thirty cycles of ten jennies were randomly assigned in one of the three groups: G0 (control group), saline; G1, 250 μg of histrelin acetate; G2, 2500 IU of hCG. Jennies were evaluated by transrectal palpation and ultrasonography, and had the administration of an ovulation-inducing agent when a follicle measuring between 29 and 32 mm of diameter was diagnosed. Jennies were monitored every 6 hours by transrectal ultrasonography until ovulation. The interval between prostaglandin administration and ovulation was lower (P < .05) in jennies from the G1 (145.2 ± 34.6 hours) and G2 (147.4 ± 27.3 hours) groups compared with the control cycle (220.0 ± 41.8 hours). Both treatments (G1, 41.15 ± 3.5 hours; G2, 37.8 ± 2.5 hours) also reduced (P < .05) the interval that jennies took to ovulate after the administration of the ovulation-inducing agent compared with the control (81.8 ± 28.8 hours). All jennies from G1 and G2 ovulated up to 48 hours after ovulation induction, whereas 100% of jennies in the control cycle ovulated later (>48 hours from the administration of saline). In conclusion, both histrelin acetate and hCG at the used dose are efficient ovulation-inducing agents in jennies promoting ovulation up to 48 hours after administration. Sao Paulo State University (Unesp) School of Veterinary Medicine and Animal Science Caxias do Sul University Sao Paulo State University (Unesp) School of Veterinary Medicine and Animal Science

Made available in DSpace on 2018-12-11T17:36:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study aimed to evaluate the antioxidant properties of coenzyme Q10 (CoQ10) during cryopreservation of semen obtained from stallions having good and bad semen freezing ability (GFA vs. BFA, respectively). Forty ejaculates (n = 20 stallions) were split into five centrifugation and five freezing extenders containing different concentrations of CoQ10 (0, 25, 50, 75 and 100 μmols/L). If CoQ10 was added to the centrifugation extender, the freezing extender had no CoQ10 added; similarly, if CoQ10 was added to the freezing extender, the centrifugation extender had no CoQ10. Semen cryopreserved on extenders containing no CoQ10 served as the control. After post-thaw total sperm motility (TM) assessments, the stallions were classified as GFA (i.e., decrease of ≤25% in TM, n = 7) or BFA (i.e., decrease of ≥40% in TM, n = 5). Stallions not fitting (n = 8) this enrollment criteria had samples discarded. After that, two straws for each extender were thawed at 37 °C for 30 s; one straw was immediately used for evaluation of sperm kinetics, plasma membrane integrity, non-capacitated spermatozoa, reactive oxygen species production, mitochondrial activity and lipid peroxidation. The second straw was kept at 37 °C for 30 min and subjected to the same assessments. Expectedly, sperm motility parameters were significantly lower for stallions with BFA. There were no effects of CoQ10 concentration or time for all parameters evaluated in the group with GFA when compared with the control extender (p > 0.05), except lipid peroxidation (p < 0.05). However, stallions with BFA had improved sperm parameters for samples processed with extenders containing CoQ10 (particularly 75 μmols/L) (p < 0.05), except for the reactive oxygen species production and mitochondrial potential (T0) in which there were no differences between the groups (p > 0.05). In summary, 75 μmols/L appears to be the optimal dose of Co-Q10, particularly, when added to the centrifugation extender. Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science Department of Veterinary Clinical Medicine College of Veterinary Medicine University of Illinois Urbana-Champaign Sao Paulo State University (UNESP) School of Veterinary Medicine and Animal Science FAPESP: 2015/25638-3

Made available in DSpace on 2018-12-11T17:33:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Persistent mating-induced endometritis (PMIE) results in decreased fertility in horses, thereby causing a significant impact in the horse market. Platelet-rich plasma (PRP), a modulator of the inflammatory response, has been largely used in veterinary medicine. Here, we investigated the effects of PRP on uterine inflammation, conception rate, endometrial polymorphonuclear neutrophil (PMN) migration, and COX-2 protein levels in the endometrial tissue. Thirteen PMIE-susceptible mares were used for artificial insemination (AI). The mares were inseminated with fresh semen in three consecutive cycles in a cross-over study design. The following cycle classifications were used: control cycle, no pharmacological interference; pre-AI, 20 mL of PRP was infused 24 h before AI; and post-AI, 20 mL of PRP was infused four h after AI. Follicular dynamics were monitored daily by transrectal ultrasound. When a follicle larger than 35 mm was detected, ovulation was induced with deslorelin acetate (1 mg, im). AI was performed 24 h after ovulation induction. Intrauterine fluid (FLU) was evaluated by ultrasonography before and 24 h after AI. PMNs in uterine cytology (CYT) and biopsy (HIS) were also observed before and 24 h after AI. Pregnancy was determined within 14 days after ovulation. Number of COX-2 positive cells was evaluated by immunohistochemistry. Both PRP treatments resulted in a decrease of PMNs in the CYT after breeding when compared to controls. FLU did not differ between cycles; however, the conception rates were significantly higher in the PRP mares. Mares positive for endometritis decreased in both treatment groups, and a more intense positive COX-2 labeling was observed in the control group when compared to the two treatment groups. In conclusion, PRP beneficially reduces inflammatory response in PMIE mares independent of when treatments were administered, thus increasing chances of successful pregnancy. Department of Animal Reproduction and Veterinary Radiology São Paulo State University – UNESP Santo Amaro University UNISA Severino Sombra University, Vassouras Equine Reproduction Service Department of Animal Medicine and Surgery Faculty of Veterinary Medicine Autonomous University of Barcelona Department of Animal Reproduction and Veterinary Radiology São Paulo State University – UNESP FAPESP: 2015/00150-8

Made available in DSpace on 2022-04-29T08:31:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 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. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Clinical Medicine College of Veterinary Medicine University of Illinois Urbana Champaign Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP)

As colheitas de sêmen em equinos são realizadas de forma convencional, com vagina artificialfechada, em que todas as frações se misturam. No entanto, a colheita de sêmen de formafracionada, separa as diferentes frações do ejaculado, cada uma dessas frações possuem umamaior ou menor quantidade de plasma seminal, sendo as glândulas sexuais acessóriasresponsáveis por sua produção. Cada garanhão possui características individuais com relaçãoà manutenção das células espermáticas durante a criopreservação, essas características podemestar relacionadas à produção do plasma seminal, assim como dos vários componentes. Sabese que existe um efeito deletério desempenhado pelos componentes do plasma seminalproduzido principalmente pelas vesículas seminais, aos espermatozoides durante o processode criopreservação, porém, esses fatores ainda são desconhecidos. Várias hipóteses sãosugeridas, tais como, a redução na produção de lipídeos e os tipos de proteínas e íonspresentes em cada uma das frações do ejaculado. Sabendo disso, a colheita fracionada podetrazer benefícios aos espermatozoides, de forma que as primeiras frações não tenham contatocom o plasma seminal produzido pelas vesículas seminais, possibilitando uma melhormanutenção espermática durante a criopreservação.

Made available in DSpace on 2018-12-11T17:32:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-01 Morphological analysis of the sperm cells were accomplished from 19 stallions comparing six different techniques (DIC, Karras, Eosin-Nigrosin, Dip Quick 5′, Dip Quick modified, Dip Quick) in order to verify the possibility of using a modification of Dip Quick stain technique to evaluate the morphology of equine semen. Data of sperm morphology were evaluated using Kolmogorov-Smirnov (KS) normality test. Parametric continuous data were compared for ANOVA followed by the Tukey test and non-parametric dates, for Kruskal-Wallis test followed by the Dunns test. Significant differences were considered when P .05) from other techniques to morphological assessment. However, the usual Dip quick technique showed to be less efficient (P

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.

Made available in DSpace on 2018-12-11T17:17:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01 The donkey jack sex glands are larger than stallions, responsible for producing most part of seminal plasma and the second fraction of ejaculate, along with epididymis tail. Plugged ampullae occur by sperm accumulation obstructing the lumen, inducing decrease in sperm quality and may cause azoospermia. In this study, a Pêga breed donkey jack, aging 4 years, was evaluated for breeding soundness evaluation due to a sudden decrease in semen parameters and low fertility rates. Palpation, measurements, and ultrasound examinations of testicles were normal; however, rectal palpation revealed increased volume of ampullae and deferent duct, and the transrectal ultrasonography revealed distended ampullae with hyperechogenic material in the ampullae lumen. After ampullae massage, the semen was collected with artificial vagina for evaluation, resulting in high concentrated semen (1.46 × 109 spermatozoa/mL) with low motility (5%), 14% of major defects, and 57% of minor defects. Plugged ampullae were suggested, and the treatment was performed by ampullae massage per rectum and three consecutive semen collections associated with the parenteral use of oxytocin 20 IU iv, aiming to discharge the semen accumulation. Daily regimen of semen collection was recommended during 10 days, and after this time, semen was collected at least three times a week. The semen parameters restored to normal (80% motility) after 30 days. The donkey jack returned to the breeding season with a regimen of 3 days a week of semen collection. Department of Animal Reproduction and Veterinary Radiology College of Veterinary Medicine and Animal Science Univ Estadual Paulista (UNESP) Department of Animal Reproduction and Veterinary Radiology College of Veterinary Medicine and Animal Science Univ Estadual Paulista (UNESP)

Made available in DSpace on 2020-12-10T19:47:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-26 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 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. Sao Paulo State Univ, Dept Anim Reprod & Vet Radiol, Coll Vet Med & Anim Sci, Botucatu, SP, Brazil Sao Paulo State Univ, Dept Anim Reprod & Vet Radiol, Coll Vet Med & Anim Sci, Botucatu, SP, Brazil

Made available in DSpace on 2019-10-06T17:13:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-15 Non-steroidal anti-inflammatory drugs (NSAIDs) are a therapeutic option for the treatment of inflammation. However, negative effects of non-selective NSAIDs for treatment of mares with endometritis have been described, including delayed uterine clearance and impairment of ovulations. Firocoxib is a specific cyclooxygenase-2 (COX-2) inhibitor and has the ability to act in the uterus of mares. We investigated the effects of firocoxib on ovulation rate, numbers of polymorphonuclear neutrophils (PMNs), and COX-2 protein levels in the endometrial tissue of susceptible mares after insemination. Two experiments were conducted. In experiment 1, twenty mares were evaluated in two consecutive estrous cycles broken into the following groups: Control - no pharmacological interference; Treatment - mares were treated with 0.2 mg/kg of firocoxib orally. The treatment began on the day of ovulation induction, and firocoxib was administered until one day after artificial insemination (AI). Ovulation was induced with 1 mg of deslorelin acetate and the mares were inseminated 24 h after the injection. Ovulation was confirmed 48 h after induction, and embryos were collected eight days after ovulation. Experiment 2: Nine mares susceptible to persistent mating-induced endometritis (PMIE) were artificially inseminated. The mares were examined with ultrasound and inseminated with fresh semen in two consecutive cycles, control and treated, in a cross-over study design. The amount of intrauterine fluid was measured, and endometrial samples were collected 24 h after AI. The number of PMNs was determined by endometrial cytology and biopsy, and COX-2 labeling in endometrial samples was evaluated by immunohistochemistry. Firocoxib treatment did not induce ovulatory failure or affect embryo recovery rate in Experiment 1. In Experiment 2, firocoxib treatment reduced inflammation after AI in mares as evidenced with results regarding PMN numbers/percentage and endometrial COX-2 staining. In conclusion, the proposed treatment with firocoxib reduced endometrial inflammation in mares susceptible to PMIE after breeding, with no adverse effects. Department of Animal Reproduction and Veterinary Radiology São Paulo State University UNESP Equine Reproduction Service Department of Animal Medicine and Surgery Faculty of Veterinary Medicine Autonomous University of Barcelona LUB Breeding Farm Cesário Lange Department of Animal Reproduction and Veterinary Radiology São Paulo State University UNESP

Seminal vesiculitis in stallions reduces fertility and is often underdiagnosed. The most common cause is infection of seminal vesicles by bacteria capable of forming biofilms and a propensity for tissue persistence, for example, Pseudomonas aeruginosa. Achieving a clinical cure is challenging because of a high rate of recurrence. Systemic antibiotic therapy does not reach adequate therapeutic concentrations within the seminal vesicles; one alternative is endoscopy-guided, local antibiotic infusion into the gland lumen, with or without concurrent systemic antibiotics. Current diagnostic and therapeutic strategies for seminal vesiculitis are less than fully satisfactory, and several studies have been conducted to improve them. This review covers traditional and newer concepts regarding seminal vesiculitis, including diagnostic and treatment methods, management of stallions with this disorder, and authors' experience with clinical cases.

Made available in DSpace on 2020-12-12T02:48:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours. São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science

Made available in DSpace on 2018-12-11T17:37:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Semen cryopreservation comprises different steps, among them are the cooling and freezing rates which significantly influence the quality of thawed sperm. Different systems with variable freezing rates are used for freezing bull semen in the field, with a consequence of variable success rates. The objective of this study was to compare different systems for freezing bull semen in the field. Five cooling methods of semen and two methods for the subsequent freezing phase (5 × 2 factorial scheme) were used. Two to four ejaculates were collected from 12 bulls with an electroejaculator. The ejaculates were diluted in BotuBov® to a concentration of 50 × 106 spermatozoa/mL in 0.5-mL straws. After dilution, the straws were cooled to 5 °C in five cooling systems: TK 4000® at a cooling rate of −0.25 °C/min (R1); TK 4000® at a rate of −0.5 °C/min (R2); Minitube® refrigerator at a rate of −2.8 °C/min (R3); Botutainer® at a rate of −0.65 °C (R4), and domestic refrigerator at a rate of −2.0 °C/min (R5). After stabilization at 5 °C for 4 h, these straws were then submitted to two freezing systems: TK 4000® at a freezing rate of −15 °C/min (C1) and Styrofoam box with liquid nitrogen at a rate of −19 °C/min (C2). Sperm kinetics were evaluated by computer-assisted sperm analysis at four time points: in fresh semen, after cooling, post-thawing, and after the rapid thermal resistance test (TRT). In addition, plasma and acrosomal membrane integrity, mitochondrial potential and intracellular H2O2 were analyzed after thawing by flow cytometry. The R1, R2 and R4 cooling systems were the most efficient in preserving sperm viability, membrane integrity and intracellular H2O2. Samples frozen in the C1 system exhibited better post-thaw and post-TRT kinetics than C2 samples. In conclusion, slower cooling curves in conjunction with a constant freezing rate obtained with the programmable unit were more efficient for freezing bull semen in the field. Centro APTA Bovinos de Corte IZ-APTA Universidade Estadual Paulista FCAV/UNESP Universidade Estadual Paulista FMVZ/UNESP Universidade Federal Fluminense UFF Universidade Estadual Paulista FCAV/UNESP Universidade Estadual Paulista FMVZ/UNESP FAPESP: 2014/11304-3 FAPESP: 2014/20360-4

The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n = 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were inseminated with frozen semen from poorly fertile stallion B. Ovulations were induced with 1 mg (intramuscular) of deslorelin acetate after the observation of a greater than 35 mm follicle and uterine edema. In postovulation insemination group, mares were inseminated once with 800 × 10(6) total sperm in a maximum 6-hour interval after ovulation. In FT group, mares were inseminated twice with 400 × 10(6) total sperm, 24 and 40 hours after induction. Mares were ultrasonographically examined for IUF accumulation 24 hours and for pregnancy diagnosis 14 days after the last insemination. Although IUF accumulation was more evident in mares inseminated once postovulation, pregnancy rates were similar for both protocols, regardless of the stallion, although a significant effect of the stallion was observed. These results indicated that FTs may be used for both highly and poorly fertile stallions as a practical tool to help spreading the use of frozen semen in equine reproduction programs.

The penis and prepuce of the stallion have a high bacterial load on its surface, forming a natural microbial flora that contaminates the semen during ejaculation. Bacterial growth in semen may cause a decline on sperm quality, viability, and fertility and predisposes the occurrence of endometritis in inseminated mares. Thus, the aim of this study was to evaluate the effect of penile wash before semen collection, the addition of different commercial skim milk–based extenders containing antibiotics (BotuSemen and INRA96), and the removal of seminal plasma by filtration on the quality, viability, and bacterial proliferation on fresh and cooled stallion semen. Animals that were never submitted to penile wash before semen collection tended to have lower bacterial contamination in the ejaculate. Semen samples extended in BotuSemen showed superiority in total motility, progressive motility, average path velocity, and rapid sperm and lower bacterial contamination in relation to semen samples extended in INRA96 after 24 hours of cooling. No difference was found in these parameters between the storage temperatures (5°C and 15°C). Furthermore, the removal of seminal plasma by filtration reduced the bacterial load in semen after cooling. In conclusion, the penile wash before semen collection tended to reduce the bacterial growth in fresh semen. The use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration were effective in reducing the bacterial contamination and preserved the quality of cooled stallion semen.

The aim of the present study was to compare the viability and fertility of bovine semen diluted in Botu-Bov (BB) commercial extender with and without the cryoprotectant glycerol then cooled at 5 degree C for 24 hours in the Botu-Flex passive cooling system and of semen diluted in BB with glycerol then frozen. One ejaculate of 30 Nelore Bos Taurus indicus bulls between 24 and 30 months of age was used for in vitro analysis. Sperm kinetics and cell viability were analyzed using computer-assisted sperm analysis and flow cytometry, respectively. Three Nelore bulls approximately 30 month old were used for in vivo test using fixed-time artificial insemination for the fertility analysis. The ejaculates were divided into three experimental groups: semen in BB extender with 7% glycerol cooled at 5 °C for 24 hours (cooled semen with cryoprotectant), semen in BB without glycerol cooled at 5 °C for 24 hours (cooled semen without cryoprotectant), and semen diluted in BB with 7% glycerol then subsequently frozen rather than cooled (frozen semen). For the fertility analysis, 762 Nelore cows (B taurus indicus) were randomly inseminated using fixed-time artificial insemination. For the groups corresponding to cooled semen with cryoprotectant, cooled semen without cryoprotectant, and frozen semen, 278, 268, and 216 cows were inseminated, respectively, and the resulting conception rates were 51% a, 44%ab and 41%b (P < 0.05), respectively. In conclusion, the fertility rates improved, when samples were cooled with glycerol at 5 °C for 24 hours compared with the frozen samples.

Summary The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation.

Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5°C for 48h using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov(®) (BB)] and another composed of 1% soy lecithin [Botu-Bov(®)-Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48h for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/10(8) cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n=253) or semen cooled for 48h in TRIS-R (n=233), BB (n=247) or BB-L (n=240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48-h of the refrigeration period (P

An incompetent vulvar seal leads to reproductive failure, and a surgical intervention might be required. The present paper describes modifications to Pouret's surgery. We suggest the use of a simple interrupted vertical mattress suture, which avoids seroma. Eighteen Brazilian Jumping Horse mares, older than 20 years and barren for 3-5 consecutive years, underwent modified Pouret's surgery. A horizontal skin incision of 3-4 cm was made half way between the anus and upper commissure of the vulva. The submucosal and connective tissue were dissected, and the rectovaginal shelf was split horizontally by sectioning the muscular and ligamentous connections between the anus, vulva, caudal portion of the rectum, and vagina until the vulva was oriented vertically. The wound was changed from a horizontal plane to a vertical plane by placing the suture vertically using approximately eight interrupted U sutures distributed in two layers with polyamide thread. The modified Pouret's surgical technique provided a perfect coaptation of the vulvar lips and a correct perineal position. Those mares that presented with horizontally tipped vulvar lips due to advanced age and stretching of the pelvic tissues by multiple foaling had their vulvas replaced. Also, the surgical procedure was easy to perform. As to fertility, of the 18 initial mares, 14 were inseminated, and all became pregnant. Thus, it was possible to conclude that the anatomical changes performed throughout the surgical procedure predisposed to a better vulvar coaptation, correcting the pneumovagina.

The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation and consequently improve frozen semen quality and fertility.

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm.

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 timepointof the cooling process. In conclusion, centrifugation, even at high speeds, did not affect goat sperm integrity and functionality when submitted to refrigeration process. 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 deespermatozoides 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.

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5°C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5°C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computer-assisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:27:28Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:27:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-01 Evaluation of the damage caused by the sperm preservation process is crucial to improving fertilization rates. The objective of this study was to evaluate the effects of refrigeration temperature (5°C and 15°C) and storage time (0, 12, 24, 48, and 72 hours) on apoptotic markers in equine semen. Membrane phosphatidylserine translocation index, caspase activation index, and DNA fragmentation index were analyzed using epifluorescence microscopy. Analysis of variance was used for statistical analysis, and Tukey test was used to compare means. The significance level was set at P < .05. The results demonstrated that for transport duration shorter than 24 hours, semen quality was maintained when stored at either 5°C or 15°C. A storage temperature of 5°C should be used when it is necessary to transport semen for longer than 24 hours. There was a significant decrease in semen quality after 48 hours of refrigeration. © 2013 Elsevier Inc. Department of Animal Reproduction and Veterinary Radiology College of Veterinary Medicine e Animal Science University of Sao Paulo State, Botucatu, São Paulo

This study aimed to determine whether deslorelin acetate could induce double ovulation in mares. In Experiment 1, eight mares were treated with prostaglandin on Day 8 (D8) after ovulation, then treated with saline or with 100 μg of a controlled-release formulation of deslorelin acetate vehicle intramuscularly (IM) every 12h from D8 after ovulation until at least two follicles reached 33 mm. At this time, ovulation was induced with 2500 IU of hCG. Artificial insemination was performed 24h after induction, and embryos were collected on the eighth day after ovulation was first detected. In Experiment 2, 112 estrous cycles in 56 mares were studied. In this experiment, the deslorelin acetate protocol was initiated only in mares that achieved a follicle with a diameter of at least 25 mm and at least one second follicle with a diameter≥20mm was detected, at which time 100 μg deslorelin acetate or saline was administered IM every 12h. The other procedures were similar to those described in Experiment 1. The variables studied were analyzed using Student's t-test and Fisher's exact test. In Experiment 1, only two mares in deslorelin group having second follicles of 20-25 mm on responded with double ovulation. In the second experiment, 82% of treated mares responded with double ovulation, and the embryo recovery per estrous cycle was 1.12 and 0.57 in the group treated with deslorelin acetate and the control group, respectively (P

Made available in DSpace on 2013-09-27T14:54:19Z (GMT). No. of bitstreams: 1 WOS000314513900032.pdf: 641496 bytes, checksum: f47cabfb2f9be059948fdda57e776789 (MD5) Previous issue date: 2012-01-01 Made available in DSpace on 2013-09-30T18:29:08Z (GMT). No. of bitstreams: 1 WOS000314513900032.pdf: 641496 bytes, checksum: f47cabfb2f9be059948fdda57e776789 (MD5) Previous issue date: 2012-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:42:18Z No. of bitstreams: 1 WOS000314513900032.pdf: 641496 bytes, checksum: f47cabfb2f9be059948fdda57e776789 (MD5) Made available in DSpace on 2014-05-20T13:42:18Z (GMT). No. of bitstreams: 1 WOS000314513900032.pdf: 641496 bytes, checksum: f47cabfb2f9be059948fdda57e776789 (MD5) Previous issue date: 2012-01-01 Sperm cryopreservation success depends upon the maintenance of spermatozoa fertility potential. Sperm cells must preserve both integrity and functionality of several cell structures. The stabilization phase must allow the exit of water from the sperm cells via osmosis. This study aimed to compare the effect of refrigeration in the commercial refrigerator (CR) and the transport/refrigeration box (TRB) upon the viability of frozen bull sperm diluted in three different extenders (A, B and C). Ten Nellore bulls, Bos taurus indicus maintained in Artificial Insemination Center were used and the spermatozoa samples was assessed for Plasma Membrane Integrity and CASA evaluation. The stabilization phase (5 degrees C/4 hours) was performed in the CR as well as in the TRB, and then samples were exposed to nitrogen vapor during 20 minutes and then plunged into nitrogen. The statistical analysis was done using the variance analysis and the significance level was set at 5%. In the CR the post-thawing parameters for PM and ALH were higher (p < 0.05) in the extender A (glicine egg-yolk) and extender B (glicine egg-free) when compared with extender C (TRIS egg-yolk). As for BCF, STR and LIN, the parameters were higher (p < 0.05) in extender B than in C. Samples that were stabilized in the TRB presented higher post-thawing parameters (p < 0.05) for PM and LIN in extender A and extender B when compared with C. BCF and STR parameters were higher (p < 0.05) in extemder B when compared with C. Extender B samples had higher (p < 0.05) PMI when stabilized in CR. The findings in this experiment enable us to say that both CR and TRB were effective in keeping the viability of post-thawing bull semen. Univ Oeste Paulista, UNOESTE, Presidente Prudente, SP, Brazil Univ Oeste Paulista, Presidente Prudente, SP, Brazil Univ Estadual Paulista, UNESP, Botucatu, SP, Brazil UNESP, Botucatu, SP, Brazil Univ Estadual Paulista, UNESP, Botucatu, SP, Brazil UNESP, Botucatu, SP, Brazil

Removal of excess seminal plasma is sometimes necessary to increase the quality and the longevity of cooled equine semen; moreover, this procedure is an indispensable step aiming to concentrate the sperm cells before freezing equine semen. Typically, the removal of seminal plasma is achieved by centrifugation; however, studies have shown that the force and duration of centrifugation can damage sperm cells and reduce the sperm recovery rate. Recently, new methodologies, such as cushion and filtration, have been described that aim to decrease the mechanical damage of centrifugation to sperm cells. This study aims to compare different methods for concentrating stallion semen.

The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment 1, sperm frozen in BC and BCLS45 exhibited similar (P>0.05) percentages of total motile sperm (61% and 61%, respectively); progressively motile sperm (27% and 27%, respectively) and sperm with intact plasma membranes (IMP; 53% and 57%, respectively). Similarly, sperm frozen in BC or BC containing any concentration of soybean lecithin maintained similar (P>0.05) percentages of total motile sperm (61-68%) and progressively motile sperm (27-31%). In the first fertility trial, we used cryopreserved semen from a single stallion was inseminated into mares. The semen from the sperm that were frozen in BC diluent resulted in a higher fertility rate (66%, 16/24) compared to the sperm that were frozen in BCLS45 diluent (17%, 5/29; P

Although several methods of testicular biopsy have been proposed previously, testicular fine needle aspiration (FNA) has proved to be the simplest, the most rapid, inexpensive, and overall the least invasive technique for obtaining testicular biopsies. Testicular FNA is indicated for fertility investigations in stallions with oligozoospermia or azoospermia. It is also used for differential diagnosis of testicular enlargement. After sedation, the stallion’s testis is punctured to obtain testicular parenchyma samples containing cells mainly from the seminiferous epithelium. The material obtained is used to perform smears which are analyzed for identification and quantification of germ cells and Sertoli cells. The results are based on the presence of the cell types found in the smears and the proportions of Sertoli cells per germ cells. In addition to being a very useful diagnostic tool, testicular FNA is also used for follow-up examinations, as it is minimally invasive.

Background: Several reproductive diseases can prevent ejaculation by the traditional method of collection. Neoplasias as squamous cell carcinoma is the most common tumor of the external genitalia of horses and its lesions usually prevent copulation. The pharmacological induction of ejaculation is an important alternative technique to obtain and preserve the genetic material of stallions incapable of ejaculating by traditional methods of semen collection. However, the protocols currently used have shown questionable results and new protocols are needed in order to increase the success rates. The aim of this study is to report the success of a new protocol in inducing ejaculation when oral imipramine and intravenous oxytocin and detomidine were administrated in a Crioulo stallion.Case: A 9-year-old Crioulo stallion was admitted at the Veterinary Hospital of the São Paulo State University, FMVZUNESP, Botucatu, Brazil, with a history of a mass located on the glans and body of the penis. The histopathological exam confirmed the diagnostic of Squamous cell carcinoma and penectomy was performed. After 10 days of surgery the stallion was submitted to 5 different protocols with 3 days interval between the follow protocols: Imipramine+Xylazine; Imipramine+ Xylazine+Oxytocin; Imipramine+Detomidine and Imipramine+Detomidine+Oxytocin.Discussion: The traditional protocol of pharmacologically-induced ejaculation with imipramine hydrochloride (3 mg/kg/v.o) and xylazine hydrochloride (0.66 mg/kg/iv) was not successful even when oxytocin (20 UI/iv) was added to this protocol. Administration of imipramine hydrochloride (3 mg/kg/v.o) two hours prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) also did not result in ejaculation. However, administration of imipramine hydrochloride (3 mg/kg/v.o) 2 h prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) associated with oxytocin (20 U.I/i.v) resulted in ejaculation. The stallion was submitted to three seminal collections with a three-day interval between administration of the protocol and ejaculated in all the attempts after approximately 5 min of detomidine and oxytocin injection, presenting mean values of 50 mL of total volume (TV) and concentration of 80x106 spermatozoa/mL. The VT was higher and concentration was lower when compared to ejaculates obtained by pharmacological induction in previous studies, probably due to daily stimulation with estrus mare to induce penile exposure in order to allow antisepsis of the surgical wound. Thus, it is believed that the large amount of total volume of this stallion is due to the high production of gel by the accessory sex glands, with consequent reduction of ejaculate concentration. The sperm kinetics were evaluated by the computerized method CASA (HTMA-IVOS-12) with total motility (MT) of 84% and progressive (MP) of 38%, with 70% of rapid spermatozoa (RAP), being considered normal to the equine specie and similar to those observed by other authors in pharmacolocally-induced ejaculates. Post-thaw sperm kinetics presented 42% of MT, 21% of MP and 28% of RAP probably due to an intrinsic sensitivity of the stallion to the freezing process. Thus, this report concludes that the protocol associating imipramine, detomidine and oxytocin was efficient in the pharmacological induction of ejaculation, presenting normal and characteristic sperm parameters of the specie. Fresh and refrigerated semen presented good parameters for use in conventional artificial inseminations while frozen semen is indicated for deep horn inseminations or for use in intracytoplasmic sperm injection (ICSI) programs.

The aim of this study was to compare the efficiency of the intravaginal (IVAI) vs. intrauterine artificial insemination (IUAI) using frozen-thawed sperm in the domestic cat. Semen was collected from two tom cats using an artificial vagina and samples were assessed for motility (computer-assisted sperm analysis (CASA)), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP/YOLK (4% of glycerol), sperm samples were loaded into 0.25 mL straws (25 x 10(6)motile sperm/straw), incubated at 5 degrees C for 20 min and cryopreserved over liquid nitrogen (LN(2)) vapor for 15 min and then immersed in LN(2). For each AI, four straws from the same male were thawed (12s at 46 degrees C) and centrifuged at 250 x g for 8 min to pellet the sperm. The supernatant was discarded and sperm pellet resuspended with the remaining liquid, approximately 100 microL, and analyzed as described above. Queens were treated with a single im injection of 100 IU eCG to induce ovarian follicular development. Final oocyte maturation and ovulation was induced with 100 IU hCG given im at 82-84 h after eCG administration. Thirty hours after hCG administration, females were inseminated either intrauterine (n=8 queens) or intravaginally (n=8 queens), using thawed sperm from a single male. Although a pronounced decrease in sperm motility, acrosome and plasma membrane integrity was observed in sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method compared with 0% pregnancy when inseminated intravaginally.

Inseminations with frozen–thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 °C for 24 h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio ® , EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio ® than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen–thawed epididymal sperm showing that Botu-Crio ® was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp + Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio ® was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.

The authors report a rare case of bilateral Leydig cell tumor in a noncryptorchid stallion, describing the gross and microscopic findings. An 8-year-old Appaloosa stallion was examined for a unilateral enlargement of the testis associated with discomfort during palpation and signs of colic. General health conditions were good. Fine-needle aspiration cytology of the testis provided the diagnosis of bilateral Leydig cell tumor. Bilateral orchiectomy was performed and the testes were submitted to histopathologic examination that confirmed the diagnosis of Leydig cell tumor.

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio ® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.