Epididymal tail solid-surface vitrification as an effective method for domestic cat sperm cryobanking.

This study aimed to describe the viability of domestic feline spermatozoa after epididymal tail vitrification. For this, 10 pairs of testis-epididymis complexes were used. The epididymal tails were vitrified using the solid-surface vitrification (SSV) method, in which two vitrification media containing ethylene glycol (EG) 40% or glycerol (GLY) 40% were tested. Vitrification with the presence of EG resulted in better results for all sperm motility parameters (motility, vigour and CASA) compared with GLY (P < 0.05). There were no statistical differences for sperm viability and acrosome integrity, plasma membrane integrity, or overall health of morphologically normal sperm before or after vitrification among experimental groups. In conclusion, epididymal tail vitrification appears to be a suitable method for long-term storage of cat sperm, especially if the procedure is performed with EG as the cryoprotectant.

In vitro assays for evaluating phytate degradation in non-ruminants: chances and limitations.

The positive effects of phytases on the environment, animal welfare and animal feed costs have resulted in the continuous development and improvement of these enzymes in the non-ruminant feed market. To test the efficacy of these phytases, a large number of experimental animals are necessary, antagonising the animal welfare aspect of these enzymes. In the present review, we summarise the most prominent available in vitro assays for evaluating phytase enzymes and how far they can reduce the number of in vivo experiments. Several in vitro assays exist that differ in their setup, extent and conditions depending on the animal of interest and the research question. With the in vitro assays described, it is not possible to fully replace in vivo trials. However, for the investigation of phytase effects in feedstuffs, the use of an in vitro assay has several advantages. In vitro assays have the potential to be used for ranking feed enzymes and as screening tools. Applying in vitro protocols will result in a reduction in the number of animals or treatments usually necessary for an in vivo trial, thus acting towards the three Rs. © 2020 Society of Chemical Industry.

Managing embryonic and calves losses after twin pregnancies induced by transfer of in vitro-produced Nellore embryos.

Two farms applying reproductive technology for the Nellore beef cattle were selected. Both farms had the same technology programme of oestrous synchronization and embryo transfer, but management was different, especially regarding twins pregnancies. In the present study, we followed the farms from the moment of oestrous synchronization, embryo transfer (two per cow), until delivery and first care of the calves. In farm A, cows presenting twin pregnancies (5 from 13) were submitted to delivery induction, as well as calves and cows were monitored after birth. In farm B, such management was not followed with the twin pregnant cows (31 from 49). In both farms, freemartinism was detected, but this was not a problem as none of the animals would be selected for breeding. No dystocia was observed in farm A, while 48% of the twin pregnancies in farm B ended up in dystocia. Furthermore, the mortality rate of new-born calves in farm A was 10%, while in farm B it reached 32%. Although twin pregnancies remain a concern, we showed here that proper management during and after delivery minimizes animal and economic losses.

Micromorphological and ultrastructural description of spermatozoa from squirrel monkeys (Saimiri collinsi Osgood, 1916).

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

Effects of a feed additive blend on broilers challenged with heat stress.

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20-22°C (control - normothermic) or to 35-39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend. RESEARCH HIGHLIGHTS Jejunum is the most sensitive intestinal segment during heat stress. Heat stress affects the expression of tight junctions and nutrient transporters. Feed management helps to alleviate the disturbances caused by heat stress. A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

B-mode ultrasonographic evaluation of long bones in Falconiformes and Strigiformes birds.

Bone fractures in birds are usually diagnosed with the aid of traditional radiography. However, this technique remains limited because of the difficulties associating this examination with real-time procedures. The aim of this study was to describe the use of B-mode ultrasound to assess the long bones of two avian orders. For the study, we used carcases of birds from the orders Falconiformes (n = 9) and Strigiformes (n = 12), with weights ranging from 108 to 1020 g. An ultrasound device with a 5-12 MHz linear probe was employed to produce images of the long bones (humerus, radius, ulna, femur and tibiotarsus). Ultrasound (US) measurements and physical measurements using a caliper were applied to compare the diameter of the bones. Images were also recorded from the US examination performed in two live patients attending the hospital with suspected bone fractures. No statistical difference was found between the two methods of measurement in carcases weighing up to 267 g (P > 0.01). The US examination provided relevant clinical information about the bone cortex and assisted in real-time surgical procedures. RESEARCH HIGHLIGHTS Long bones of Falconiformes and Strigiformes birds were assessed with B-mode ultrasound. Ultrasound analysis is a relevant tool in clinical orthopaedics for avian species. Ultrasound of the bone might be applied for monitoring of healing processes.

Cryosurvival after exposure of IVF-derived Nellore embryos to different cryoprotectants and exposure times during vitrification.

We evaluated the effects of the vitrification solution (i.e., ethylene glycol (EG) + dimethyl sulfoxide (DMSO) with or without propylene glycol (PG)) and of exposure time on the re-expansion and hatching rates of vitrified Bos indicus embryos. In vitro produced embryos (n = 1050) were divided into seven groups: control group (non-vitrified embryos) and six vitrification groups with different cryoprotectant concentrations and exposure times. After vitrification, embryos were cultured for determination of re-expansion and hatching rates. Vitrification with 25% DMSO +25% EG (exposure for 1 min and 20 s) resulted in the highest re-expansion (65.2%) and hatching (68.2%) rates. The lowest re-expansion and hatching rates were observed in vitrification with 12.5% DMSO + 25% EG + 12.5% PG with both tested exposure times (i.e., 3 min + 1 min and 1 min + 20 s). A combination of DMSO + EG is efficient to preserve blastocysts, especially following a short exposure time.

Vitrification of domestic cat (Felis catus) ovarian tissue: Effects of three different sugars.

We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.

Unilateral ovarian absence in a Black-headed Squirrel Monkey (Saimiri vanzolinii Ayres, 1985), a threatened neotropical primate species.

Ovarian agenesis is an unusual anomaly with traumatic or congenital origin. In the present case report, we describe our findings in a senile S. vanzolinii female. As this neotropical primate species is listed as vulnerable, with limited geographic distribution in the Brazilian Amazonia, ovarian agenesis may be an important finding to be reported.

Seminal coagulation and sperm quality in different social contexts in captive tufted capuchin monkeys (Sapajus apella).

In the present study, we aimed to assess the influence of different social contexts on the seminal coagulation and sperm quality in captive tufted capuchin monkeys. For this, males were housed either individually, in mixed-sex groups (with females), or in male-only groups. Monkeys were housed in cages and each cage type (i.e., individual or group cage) was placed in a different room. Forty-one males were subjected to semen collection by rectal electroejaculation. The degree of seminal coagulation was determined on a scale of I-IV. Seminal volume, sperm concentration, sperm motility, vigor, and plasma membrane integrity were evaluated for all ejaculate samples. All ejaculates collected showed degrees of coagulation between II and IV, where the majority presented coagulation degree IV, when collected from animals housed in groups. No statistical differences among percentages of coagula degree when samples were collected from males housed individually. Animals housed in group cages (male-only groups and mixed-sex groups) showed a significantly higher percentage of ejaculates at degree IV than males housed individually. Seminal volume was not affected by the coagula degree but by the housing system, where animals housed individually showed the highest volume (543 µl) when compared with those animals from male (273 µl) and mixed-sex (318 µl) groups. No differences were observed in semen volume when comparing male-only groups with mixed-sex groups. Sperm motility was affected by both housing system and coagula degree. Samples with coagula degree IV from animals housed individually showed the highest (72%) sperm motility percentages. Sperm plasma membrane integrity was lower when samples were presenting coagula degree II + III and collected from male- (17%) or mixed-sex (23%) groups. However, this housing system effect was not observed when sperm was obtained from coagula degree IV semen. Sperm vigor was neither affect by housing system or coagula degree.

Mitotic index and morphological characteristics of ovarian small follicles from goats submitted to nutritionally unbalanced regimens.

The objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding-refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

Population estimate and morphometry of ovarian preantral follicles from three recently recognized squirrel monkey species: a comparative study.

We describe morphological and morphometrical characteristics of preantral ovarian follicles from three recently recognized Saimiri species: S. macrodon, S. cassiquiarensis and S. vanzolinii; the last one a threatened species. Ovaries from four adult monkeys were evaluated: one pair from a pregnant S. macrodon, two ovarian pairs from S. cassiquiarensis females (one of them pregnant), and one left ovary from a senile S. vanzolinii, applying classical histology. Follicular preantral population was quantified and morphology and morphometry of primordial, primary and secondary follicles were evaluated. Follicular preantral population varied among species, being 347,153 in the ovaries of the S. macrodon, 270,342 and 278,376 in the ovaries of both adult non-pregnant and pregnant S. cassiquiarensis females, and 28,149 in the ovary from a senile S. vanzolinii. Most follicles were at primordial or transition stages, except for the senile S. vanzolinii female, which presented the lowest percentages of primordial and transition follicles when compared with primary and secondary ones. Most preantral follicles (>70%) were morphologically normal in the ovaries from all studied S. macrodon and S. cassiquiarensis females, but the ovary of the senile S. vanzolinii female presented a significant decrease in the percentage of normal follicles (primordial: 61%, transition: 52%, primary: 54%, and secondary: 48%). In general, follicular diameter increased significantly from primordial to transition, and subsequently from primary to secondary follicles.

Extender supplementation with catalase maintains the integrity of sperm plasma membrane after freezing-thawing of semen from capuchin monkey.

We aimed to evaluate the effect of supplementation of ACP-118® extender with the antioxidant catalase (10 and 50 µg/ml) on Sapajus apella sperm motility, vigour, and plasma membrane integrity during the processes of seminal liquefaction, cooling, and freezing. Catalase did not affect any of the evaluated parameters after semen dilution or cooling. Cryopreserved sperm in the presence of 50 µg/ml catalase presented a plasma membrane integrity similar to that fresh sperm, however.

Morphologic analysis of sperm from two neotropical primate species: comparisons between the squirrel monkeys Saimiri collinsi and Saimiri vanzolinii.

Sperm morphometry can be applied to identify different animal groups and species and to evaluate sperm quality. Furthermore, knowledge on species-specific differences will help to enhance biological information, as well as to develop efficient reproductive technologies. The aims in the present study were to describe sperm morphometry from the recently characterized species S. collinsi and S. vanzolinii, to verify if the morphometric sperm patterns are similar or different between both species, and to determine if the sperm morphometry is affected by the levels of sperm defects using the S. collinsi as a model. Semen was collected from S. collinsi (n = 10) and S. vanzolinii (n = 2) monkeys, and sperm was submitted to morphological analysis. From the 10 samples from S. collinsi, five presented sperm of poor quality and two subgroups were formed for this species, i.e. high and poor quality sperm. Data on sperm motility and vigour were analysed, as well morphometric parameters on sperm head and tail. It was observed the normal morphometry was correlated with high quality sperm. Poor quality sperm presented smaller and 7% more ellipticity in their head, when compared with high quality sperm. Sperm from S. vanzolinii presented larger head than those from S. collinsi, but tail lengths were similar. Sperm morphometry can be used as a complementary tool to predict sperm motility and vigour for the S. collinsi species, and S. collinsi appear as a suitable model for S. vanzolinii.

Cooling and freezing of sperm from captive, free-living and endangered squirrel monkey species.

Germoplasm banking is an important tool for the preservation of genetic material from Neotropical primates in captivity, and from free living species, especially the endangered ones like Saimiri vanzolinii (Black-headed squirrel monkey), a primate with a low incidence area (870 km(2) of floodplains) in the southern part of the Mamirauá Sustainable Development Reserve, Brazil. Therefore, in the present study we aimed to develop a sperm cryopreservation protocol comparing sperm cooling in presence (T1) and absence (T2) of egg yolk, and to test freezing protocols to preserve semen from captive (Saimiri collinsi), and free-living (Saimiri vanzolinii, Saimiri cassiquiarensis and Saimiri macrodon) New World primates. Cooling preserved sperm of S. collinsi in all evaluated microscopic parameters, except for sperm motility. No differences were observed among the treatments, indicating that semen of this species can be cooled without egg yolk. Freezing did not affect sperm quality of S. collinsi, except plasma membrane integrity that was negatively affected. Generally, a good maintenance rate was observed between cooling and thawing of semen for the four species, showing the positive translational application of protocols from S. collinsi to the free-living species. Developed freezing protocol proved to be useful for sperm cryopreservation of S. collinsi and in field conditions.

Cinnamaldehyde, Carvacrol and Organic Acids Affect Gene Expression of Selected Oxidative Stress and Inflammation Markers in IPEC-J2 Cells Exposed to Salmonella typhimurium.

Essential oils and organic acids are used as feed additives to improve health status and reduce colonization with pathogens. Although bactericidal in vitro, concentrations achieved in the animal gut are probably not lethal to pathogens. The aim of this study was to investigate the effects of cinnamaldehyde, carvacrol and cinnamic, lactic and propionic acids on the ability of Salmonella typhimurium ATCC 14028 (ST) to invade intestinal epithelial cells (IPEC-J2) and on the expression levels of immune related genes in the cells. The minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) were determined and influence on the invasion capacity of ST was investigated. The structure of fimbriae and flagella was analysed by electron microscopy, and expression levels of HSP70, IkBa, IL-8 and IL-10 in the IPEC-J2 cells were carried out by q-PCR. Cinnamaldehyde, carvacrol and cinnamic and propionic acids inhibited ST invasion but not cell viability, bacterial viability and motility or the development of flagella. Propionic acid and cinnamaldehyde in combination with cinnamic acid caused structural impairment of fimbriae. Cinnamaldehyde up-regulated expression of HSP70 irrespective of the presence of organic acids or ST; exposure to carvacrol induced HSP70 only in the presence of propionic acid and ST. © 2016 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.

Quantitative histo-morphometric analysis of heat-stress-related damage in the small intestines of broiler chickens.

The aim of the current research was to present a methodological approach allowing reproducible morphometric and morphological (Chiu/Park scale) analyses of the alterations in the intestines of broilers exposed to heat stress. Ross broilers were exposed over four consecutive days to a high-temperature regime in controlled climate rooms, with a day temperature of 39°C (±1°C) and a night temperature of 25°C (±1°C), respectively. A control group was kept at an ambient temperature of 25°C (±1°C) during the entire experimental period. At the end of the exposure period, the birds were sacrificed and specimens were taken of the duodenum, jejunum and ileum for histology. Blood was collected for oxidative stress analysis. Histo-morphological and morphometric analyses of the intestines indicated that the duodenum and jejunum showed more damage than the ileum. The major alterations in the control intestines were limited to the villus tips, while heat stress led to villus denudation and crypt damage. When compared with morphologically normal villi, heat-stress-associated alterations were also observed in villus height (decreased), villus breadth at base (increased) and epithelial cell area (decreased). Birds exposed to heat stress presented with an increase in glutathione peroxidase activity and a decreased antioxidant capacity. It can be concluded that the chosen model allows a reproducible quantification of heat stress effects, which is suitable for the evaluation of dietary intervention strategies to combat heat stress conditions.

Trolox enhances follicular survival after ovarian tissue autograft in squirrel monkey (Saimiri collinsi).

The aim of this study was to evaluate ovarian tissue pre-treatment with 50 µM Trolox followed by heterotopic transplantation in squirrel monkeys (Saimiri collinsi) and to assess tissue functionality via immunohistochemical analysis of the stroma and ovarian follicles. Five healthy and sexually mature squirrel monkey (Saimiri collinsi) females were used. Heterotopic autografting of fresh ovarian tissue with or without previous exposure to the antioxidant Trolox was performed and grafts were recovered for analysis 7 days later. Tissue vascularisation was confirmed by both macroscopic inspection and cluster of differentiation 31 (CD31) staining. Trolox prevented massive follicular activation and kept the percentages of morphologically normal follicles higher than in untreated grafts. Expression of anti-Müllerian hormone in developing follicles was observed only in controls and Trolox-treated grafts. Also, immunostaining for growth differentiation factor-9 was positive only in primordial follicles from controls and from Trolox-treated grafts. Although Trolox improved follicular quality and avoided apoptosis in stromal cells, ovarian tissue fibrosis was increased in Trolox-treated grafts, mainly due to an increase in collagen Type I synthesis.

Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology.

Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients' exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human.

An Algoclay-Based Decontaminant Decreases Exposure to Aflatoxin B1, Ochratoxin A, and Deoxynivalenol in a Toxicokinetic Model, as well as Supports Intestinal Morphology, and Decreases Liver Oxidative Stress in Broiler Chickens Fed a Diet Naturally Contaminated with Deoxynivalenol.

The aims of this study were (i) to determine the effect of an algoclay-based decontaminant on the oral availability of three mycotoxins (deoxynivalenol; DON, ochratoxin A; OTA, and aflatoxin B1; AFB1) using an oral bolus model and (ii) to determine the effect of this decontaminant on the performance, intestinal morphology, liver oxidative stress, and metabolism, in broiler chickens fed a diet naturally contaminated with DON. In experiment 1, sixteen 27-day-old male chickens (approximately 1.6 kg body weight; BW) were fasted for 12 h and then given a bolus containing either the mycotoxins (0.5 mg DON/kg BW, 0.25 mg OTA/kg BW, and 2.0 mg AFB1/kg BW) alone (n = 8) or combined with the decontaminant (2.5 g decontaminant/kg feed; circa 240 mg/kg BW) (n = 8). Blood samples were taken between 0 h (before bolus administration) and 24 h post-administration for DON-3-sulphate, OTA, and AFB1 quantification in plasma. The algoclay decontaminant decreased the relative oral bioavailability of DON (39.9%), OTA (44.3%), and AFB1 (64.1%). In experiment 2, one-day-old male Ross broilers (n = 600) were divided into three treatments with ten replicates. Each replicate was a pen with 20 birds. The broiler chickens were fed a control diet with negligible levels of DON (0.19-0.25 mg/kg) or diets naturally contaminated with moderate levels of DON (2.60-2.91 mg/kg), either supplemented or not with an algoclay-based decontaminant (2 g/kg diet). Jejunum villus damage was observed on day 28, followed by villus shortening on d37 in broiler chickens fed the DON-contaminated diet. This negative effect was not observed when the DON-contaminated diet was supplemented with the algoclay-based decontaminant. On d37, the mRNA expression of glutathione synthetase was significantly increased in the liver of broiler chickens fed the DON-contaminated diet. However, its expression was similar to the control when the birds were fed the DON-contaminated diet supplemented with the algoclay-based decontaminant. In conclusion, the algoclay-based decontaminant reduced the systemic exposure of broiler chickens to DON, OTA, and AFB1 in a single oral bolus model. This can be attributed to the binding of the mycotoxins in the gastrointestinal tract. Moreover, dietary contamination with DON at levels between 2.69 and 2.91 mg/kg did not impair production performance but had a negative impact on broiler chicken intestinal morphology and the liver redox system. When the algoclay-based decontaminant was added to the diet, the harm caused by DON was no longer observed. This correlates with the results obtained in the toxicokinetic assay and can be attributed to a decreased absorption of DON.