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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 2022-04-29T08:31:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Pregnancy rates after embryo transfer (ET) are disappointing in donkey species. This study aims to report two successful ET of mini-donkey embryos using Brazilian Northeastern jennies as recipients. Eighteen embryo flushes were performed 9 days post-ovulation in two non-pregnant mini-donkeys jennies (11 and 7 cycles per jenny). Eleven embryos (61%, 11/18) were collected and transferred to Brazilian Northeastern jennies 4–6 days post-ovulation by conventional (n = 6) or an alternative (n = 5) technique. The alternative method consisted of inserting a Polansky equine vaginal speculum smeared with lubricant in the vagina of the recipient jenny. The arms of the speculum were extended to allow the visualization of the cervix. Then, using an adapted crafted, elongated, toothed tissue grasping forceps, the external cervical os was held, and the cervix was gently pulled backward, aiming to straight the cervical canal. The ET gun was inserted through the vagina and cervix by visual inspection, and the embryo was released into the uterine lumen. All embryos collected were Grade 1 and classified as Expanded Blastocysts. No jennies become pregnant after conventional ET (0/6), whereas two recipient jennies (40%, 2/5) become pregnant and delivered offspring in the following year after ET using the alternative technique. In conclusion, Brazilian Northeastern jennies can be used as embryo recipients using the alternative method proposed in the present study. However, further investigations are needed to improve the knowledge and results of ET in donkey species. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University UNESP Ross University School of Veterinary Medicine Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University UNESP

Made available in DSpace on 2021-06-25T10:58:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Background: This study aimed to evaluate the inflammatory response of miniature horses subjected to open and half-closed orchiectomy by physical examination, blood cell count, peritoneal fluid evaluation, total plasma protein, fibrinogen, and serum amyloid A (SAA) concentrations. Methods: Thirteen male healthy miniature horses were divided into two groups, according to the surgical approach: half-closed technique (HCT) and open technique (OT). The HCT group was subjected to ligation of the spermatic cord followed by its sharp incision, and closure of the vaginal tunic, and the OT group was only submitted to cord ligation. Prior to, and at 1, 2, 3 and 5 days after the surgery, a general and specific physical examination, blood cell counts, total plasma protein, peritoneal fluid evaluation, fibrinogen, and SAA concentrations were performed. Results: Higher postoperative perilesional oedema, rectal temperature, and fibrinogen were observed in the HCT group. Groups did not differ as to SAA concentrations. The evaluated local and systemic inflammatory profile demonstrated that, as expected, surgery resulted in inflammation in both groups. Conclusions: The group subjected to the HCT showed a more intense and lasting inflammatory response. However, despite the different postoperative inflammatory profiles, both groups presented a favourable outcome and recovery. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Clinics School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Clinics School of Veterinary Medicine and Animal Science São Paulo State University (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 2020-12-10T20:02:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01. Added 1 bitstream(s) on 2021-07-15T15:25:06Z : No. of bitstreams: 1 S1984-31432020000100212.pdf: 471561 bytes, checksum: d2aca3275f123f4ad49cdc287feb29a8 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process. Univ Estadual Paulista, Dept Cirurgia Vet & Reprod Anim, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Bioestat, Botucatu, SP, Brazil Univ Estadual Paulista, Dept Cirurgia Vet & Reprod Anim, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Bioestat, Botucatu, SP, Brazil FAPESP: 2013/02050-5

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

Made available in DSpace on 2019-10-06T16:57:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 The aim of this study was to investigate the influence of sperm concentration, sperm quality, and semen deposition site on uterine inflammatory response in mares. Twenty-five mares with healthy reproductive tracts were used for artificial inseminations (AIs) using frozen-thawed semen and divided into 5 experimental groups: Control—insemination with 800 × 106 total spermatozoa in the body of the uterus (n = 5); G2—insemination with 40 × 106 total motile sperm (TMS) into uterine body (n = 5); G3—insemination with 40 × 106 TMS at the uterotubal junction by hysteroscopy (HI) (n = 5); G4—insemination with 40 × 106 TMS at the uterotubal junction by HI, with frozen-thawed semen selected by Percoll gradient (n = 5); G5—insemination with 40 × 106 total motility sperm into uterine body, using frozen-thawed semen selected by Percoll gradient (n = 5). Number of polymorphonuclear neutrophils in uterine cytology was observed at 8 and 24 hours after AI. A reduced inflammatory endometrial reaction (P .05) regardless of the type of insemination (conventional or hysteroscopic). In addition, mares inseminated by hysteroscopy showed a greater local inflammatory reaction (P

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