The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Ability of donkey sperm to tolerate cooling: Effect of extender base and removal of seminal plasma on sperm parameters and fertility rates in mares.

Artificial insemination using cooled-transported semen has marked importance in equine breeding programs around the world, and the high value of mules has generated avid interest in donkey semen biotechnology. However, donkey semen cools poorly in commercially available equine extenders. Therefore, this study aimed to develop approaches to improve the ability of donkey semen to tolerate cooling. Ejaculates of seven donkeys (n = 21) were cooled at 5°C for 48 h in three different extenders (milk-based, SM; sodium caseinate-based, SC; or egg yolk-based, EY) in the presence or absence of seminal plasma (centrifugation, C). Sperm motility, plasma membrane integrity (PMI), plasma membrane stability (PMS), mitochondrial membrane potential (HMMP), intracellular hydrogen peroxide (H2O2), and intracellular superoxide ( O 2 - ) were assessed before, 24 h, and 48 h post-cooling. In addition, 15 mares (163 estrous cycles) were randomly inseminated with semen from two jacks (Jack 1, n = 90; Jack 2, n = 73) previously cooled for 24 h under one of the treatments (SM, SC, EY, SM-C, SC-C, or EY-C). Groups EY, SC-C, and EY-C (P < 0.05) demonstrated superior sperm analytical parameters to SM at 24 and 48 h. Centrifugation positively affected sperm analytical parameters in cooled donkey semen extended in SM and SC (P < 0.05). Mares bred with semen extended in SC (67%, 18/27), SC-C (89%, 24/27), EY (89%, 25/28), or EY-C (74%, 20/27) had significantly greater conception rates than mares bred with SM (33%, 9/27; P < 0.05). Mares bred with SM-C had intermediate conception rates (59%, 16/27). In conclusion, SC and EY improved the cooling ability and fertility of donkey semen in horse mares, and centrifugation positively affected donkey semen extended in SM.

Use Low Ozone Dosages has Positive Effects on the Cooling and Cryopreservation of Equine Semen.

The aim of this study was to determine an ozone dosage capable of inducing pro-oxidation, and to verify its action on sperm cells during the process of cooling and cryopreservation of equine semen. In this study, we evaluated the ozone concentrations of 2µg/mL,15µg/mL, 30µg/mL e 60 µg/mL added in equine semen cooling and freezing extenders. Samples were evaluated for sperm kinetics patterns, function of sperm structures and lipid peroxidation. In the experiment, the concentration of 15 µg/mL showed higher total and progressive motility when comparing to control (60.3±3 and 40.7±3.4 vs. 54.9±4 e 35.0±4.4, respectively, P < .05) at M24 of cooling; The concentration of 2 µg/mL showed higher percentage of intact plasma and acrosomal membrane when comparing to control at M24 (51.1±3.6 vs. 46.1±3.9, P < .05), M24 after 30 minutes of incubation (43.4±3.1 versus 32.4±2.6, P <.05). The concentration of 2 µg/mL showed higher percentage of intact plasma and acrosomal membrane (P <.05) comparing to control at moments M0 (43.5±5.0 vs. 36.3±3.5), M30 (41.0±3,7 vs. 35.3±2,9) e M60 (39.0±7.0 vs. 31.4±5.4). Thus, it can be concluded that low doses of ozone can lead to a positive response in the sperm kinetics patterns and sperm structures after sperm storage at low temperatures. Higher concentrations (30 and 60 µg/mL) were harmful in the cooling and cryopreservation of equine semen.

First successful frozen semen of the maned wolf (Chrysocyon brachyurus).

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.

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

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

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility.

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.

Periovulatory administration of firocoxib did not alter ovulation rates and mitigated post-breeding inflammatory response in mares.

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.

Sexual maturity and fertility-related measures in young Nellore bulls receiving long-term dietary supplementation with rumen-protected polyunsaturated fatty acids.

The objective of this study was to evaluate the effects of long-term supplementation with rumen-protected fatty acids (FA) on growth and reproductive parameters of young Nellore bulls in a grazing regime. Forty-eight young bulls were distributed into two groups: FA (supplemented with rumen-protected polyunsaturated FA); and control (control fat-free supplement). The animals were supplemented from 14.3 to 24.6 months of age and growth and reproductive parameters were evaluated at 28-day intervals. The semen was cryopreserved in the last collection and fresh and post-thaw semen samples were evaluated. Feeding FA did not affect (P > 0.05) growth, reproductive parameters (scrotal circumference, sperm concentration per mL of ejaculate, percentage of sperm defects, sperm quality and fertility in vitro), or testicular ultrasonographic characteristics. However, thawed semen from bulls fed FA exhibited better quality (P < 0.05) than control semen for the following parameters evaluated by computer-assisted sperm analysis: average path velocity [µm/s: 90.48 vs. 79.66 post-thaw and 74.81 vs. 72.80 post-rapid thermoresistance test (TRT)], straight-line velocity (µm/s: 72.37 vs. 65.20 post-thaw and 64.96 vs. 63.25 post-TRT), and curvilinear velocity (µm/s: 148.44 vs. 131.31 post-thaw and 115.68 vs. 113.35 post-TRT). In addition, feeding FA increased peripheral concentrations of testosterone, leptin, total cholesterol and high-density lipoprotein. In conclusion, the increase in testosterone concentrations in bulls fed FA was not related to variations in growth parameters and sexual maturity. In addition, post-thawing sperm velocities were enhanced by diet, however, such increases were not related to better in vitro embryo production rates.

Effects of coenzyme Q10 on semen cryopreservation of stallions classified as having good or bad semen freezing ability.

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.

Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses / Isolamento, cultura, caracterização e criopreservação de células tronco derivadas de camada amniótica mesenquimal e de tecido do cordão umbilical de fetuses bovinos

Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.(AU)

As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação. Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. As células tronco mais utilizadas, tanto na medicina humana como na medicina veterinária são as células tronco mesenquimais derivadas da medula óssea e do tecido adiposo. Atualmente, uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (TCU) e sangue do cordão umbilical (SCU) pode ser observada, já que estas fontes podem ser colhidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA e TCU de fetos bovinos colhidos no momento do parto e de fetos do terço inicial da gestação em abatedouro-frigorífico. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e TCU no momento do parto (n=6) e de MA e TCU no terço inicial de gestação (n=6), as quais foram submetidas às análises morfológicas, imunofenotípica por imunocitoquímica e citometria de fluxo, diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e ainda, avaliação da viabilidade após a criopreservação por citometria de fluxo. Todas as amostras dos diferentes grupos demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para ou baixíssima expressão de CD34 (MA: TI-0,3%a, PA-3.4%b; TCU: TI-0,4%, PA-1.4%) e nula ou baixa expressão de MHC II (MA: TI-1.5%a, PA-9.7%b; UCT: TI-0.7%a, PA-5.7%b. Apresentaram também capacidade de diferenciação in vitro nas três linhagens mesodermais e quando analisadas pós criopreservação por citometria de fluxo, todas as amostras apresentaram viabilidade de 80%. Estes resultados indicam que MA e TCU, obtidos tanto no momento de parto como em abatedouro, de fetos bovinos podem ser utilizados como fonte não invasiva e indolor de CTMs e possibilitam a formação de bancos de armazenamento de células.(AU)