Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR.

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Temporal changes in ewe vaginal microbiota throughout gestation.

Introduction: Numerous factors are known to influence reproductive efficiency in ewes, but few studies have investigated the potential role of vaginal microbiota in sheep reproductive success. The objective of this study was to thoroughly characterize the ewe vaginal microbiota throughout the course of pregnancy. Methods: Vaginal samples were collected from 31 pregnant Hampshire and Hampshire X Suffolk crossbred ewes on a weekly basis from pre-breeding to pregnancy testing and then biweekly until just after lambing. To characterize the vaginal microbial communities, DNA was extracted and 16S rRNA gene Illumina MiSeq amplicon sequencing was performed. Results and Discussion: Alpha diversity metrics indicated an increase in species richness, evenness, and overall diversity throughout gestation. Distinct shifts in the bacterial communities were observed during gestation and were segregated into three periods: early gestation, a transitional period and mid/late gestation. During early gestation, Actinobacillus, Histophilus, and unclassified Leptotrichiaceae were found in greater relative abundance. During the transitional period, a population shift occurred characterized by increasing relative abundance of Streptococcus and Staphylococcus. During mid/late gestation, Staphylococcus, Streptococcus, and Ureaplasma had the greatest relative abundance. These shifts in the microbial population throughout the ewe's gestation are likely related to hormonal changes triggered by the growing conceptus, specifically increasing blood concentration of progesterone. The transitional period shift in vaginal microbial communities potentially aligns with the placental take-over of progesterone production from the corpus luteum at approximately day 50 after conception (gestational week 7). Understanding the observed variability of the vaginal microbiota throughout pregnancy will allow for future comparison of ewes that did not become pregnant or had abnormal pregnancies, which could lead to the discovery of potential bacterial biomarkers for pregnancy outcome; this understanding could also lead to development of probiotics to improve sheep reproductive success.

Evaluation of peripartum supplementation of methionine hydroxy analogue on beef cow-calf performance.

The objective was to evaluate the effects of peripartum supplementation of a methionine hydroxy analogue (MHA) to primiparous, spring-calving beef females on dam and progeny performance. Angus heifers (n = 60) were blocked by expected parturition date, stratified by body weight (BW) and body condition score (BCS), and randomized to 1 of 15 pens. Pens were randomly assigned to 1 of 3 dietary treatments: a basal diet supplemented with 0 (M0), 15 (M15), or 30 (M30) g/animal/d of MHA (provided as MFP feed supplement, Novus International Inc., St. Charles, MO). Diets were fed from 45 ±â€…13 (SD) d pre-calving through 81 ±â€…13 d postpartum (DPP), after which all cow-calf pairs were managed as a single group on pasture until weaning (199 ±â€…13 DPP). Dam BW, BCS, and blood samples were taken at 6 predetermined timepoints. Progeny data were collected at birth, 2 intermediate timepoints, and at weaning. Milk samples were collected for composition analysis at 7 ±â€…2 DPP and at 55 ±â€…5 DPP. Serial progesterone samples were analyzed to establish resumption of cyclicity, and ultrasonography was performed at 55 ±â€…5 DPP to evaluate ovarian function. Cows were bred via artificial insemination at 82 ±â€…13 DPP and subsequently exposed to bulls for a 55-d breeding season. Pen was the experimental unit, and preplanned orthogonal contrasts were tested (linear effect and M0 vs. M15 + M30). Dam BW and BCS were not affected by treatment (P ≥ 0.29) throughout the study. Week 1 milk fat concentration increased linearly (P = 0.05) and total solids tended to increase linearly (P = 0.07) as MHA increased; however, no other milk components were affected (P ≥ 0.16). Treatment did not affect (P ≥ 0.16) dam reproductive parameters or progeny growth from birth until weaning. Post-calving, circulating methionine equivalents tended to linearly increase (P = 0.10) with increasing MHA supplementation. At breeding, plasma urea N linearly decreased (P = 0.03) with increased supplementation of MHA, and plasma non-esterified fatty acids were less (P = 0.04) in MHA-supplemented dams compared with dams receiving no MHA. Maternal circulating glucose, glutathione peroxidase, and thiobarbituric acid-reactive substances were not affected (P ≥ 0.15) by treatment at any point. These data indicate that peripartum supplementation of MHA may increase milk fat composition shortly after calving, but MHA supplementation did not improve progeny growth or dam reproductive performance in the current study.

Ewe Vaginal Microbiota: Associations With Pregnancy Outcome and Changes During Gestation.

Reproductive performance is paramount to the success of livestock production enterprises focused on lamb meat production. Reproductive success is influenced by various factors, possibly including the reproductive tract microbial communities present at the time of copulation and throughout pregnancy. There are few publications that identify the vaginal microbial communities of livestock, and even fewer exist for sheep. To compare ewe vaginal microbial communities, vaginal swabs were taken from 67 Hampshire and Hampshire X Suffolk crossbred ewes from the Iowa State University sheep farm at a pre-breeding time point (S1) and after pregnancy testing (S2). Animals that were determined pregnant were sampled again within a few days of expected parturition (S3). DNA was extracted from these swabs, and 16S rRNA gene Illumina MiSeq amplicon sequencing was conducted to fingerprint the bacterial communities found within this system. Pre-breeding time point samples showed no differences in community structure between animals later found to be pregnant or non-pregnant, but significant changes were detected in species richness (Chao; P < 0.001) and species diversity (Shannon; P < 0.001) at the second sampling time point. A higher microbial diversity within the S2 time point samples may suggest a more stable environment driven by pregnancy, as this increased diversity is maintained in pregnant animals from the S2 to the S3 time point. Additionally, several bacterial phylotypes, such as Mannheimia, Oscillospiraceae-like OTUs and Alistipes, were more abundant at either the S1 or S2 time points in animals that established pregnancy, suggesting a beneficial effect on pregnancy outcome. This study identifies changes within the microbial communities of the ewe vagina before and during gestation and offers inferences on how these changes may impact pregnancy outcome. Information presented herein offers new knowledge about sheep vaginal microbial communities and serves as a starting point to help guide researchers to improve sheep reproductive performance in the future.

First serological evidence of BHV-1 virus in Algerian dromedary camels: Seroprevalence and associated risk factors.

Infectious bovine rhinotracheitis (IBR), caused by bovine herpesvirus-1 (BHV-1), is a major livestock health concern in many countries of the world. The objectives of this cross-sectional study were (i) to estimate the seroprevalence of BHV-1 infection and (ii) to assess risk factors associated with this disease in dromedary camels in four districts of Algeria. Blood samples were taken from 865 camels from 84 randomly selected herds, and serum was analyzed for presence of antibodies against BHV-1 by indirect enzyme linked immunosorbent assay (ELISA). Logistic regression was used to determine associations between seroprevalence and potential risk factors (collected using a questionnaire). Antibodies against BHV-1 were detected in 3.7 % (32/865) of samples. Eighteen of 84 camel herds had at least one BHV-1 seropositive camel, giving a herd seroprevalence of 21.4 %. Based on univariate analysis, the introduction of purchased animals and contact with others animal herds appeared as major risk factors. By using multivariate analysis, the only important risk factor was introduction of new animals. This study provided, for the first time, evidence of BHV-1 infection in dromedary camels in Algeria; it also provided estimates of seroprevalence of this disease and suggests that camels may serve as a reservoir of BHV-1 for spread to other species.

Impact of cria protection strategy on post-natal survival and growth of alpacas (Vicugna pacos).

The objective of this study was to evaluate the impact of different management systems on the postnatal survival and growth of alpaca crias. The study was conducted during the alpaca calving season in the Peruvian Andes. Animals were fed on native pastures; during the day they went out to graze, but at night they were brought into a corral. A total of 150 alpaca singleton neonates were randomly assigned to one of three cria protection strategies immediately after consuming colostrum. The first group consisted of 50 crias who slept in an open-corral (OC) without shelter. The second group was comprised of 50 crias fitted with body vests (BV) who stayed overnight in an open-corral without shelter. The third group spent nights in a semi-open shed (SH). Cria survival was recorded daily, and body weight was recorded weekly. Survival to 12 weeks of age was higher (P = 0.001) for BV (100%) than for SH (76%) or OC (64%) which were not different from each other. Daily body weight gain (kg/day) during the first 12 weeks of life was higher (P < 0.001) for BV (0.17 ± 0.03) than for SH (0.14 ± 0.02) or OC (0.13 ± 0.04). There was no effect (P < 0.979) of cria sex on daily body weight gain. Results of this study revealed that fitting neonatal crias with a BV is a viable management strategy to enhance cria postnatal survival and daily body weight gain.

Birth of a Live Cria After Transfer of a Vitrified-Warmed Alpaca (Vicugna pacos) Preimplantation Embryo.

The alpaca (Vicugna pacos) is an important species for the production of fiber and food. Genetic improvement programs for alpacas have been hindered, however, by the lack of field-practical techniques for artificial insemination and embryo transfer. In particular, successful techniques for the cryopreservation of alpaca preimplantation embryos have not been reported previously. The objective of this study was to develop a field-practical and efficacious technique for cryopreservation of alpaca preimplantation embryos using a modification of a vitrification protocol originally devised for horses and adapted for dromedary camels. Four naturally cycling non-superovulated Huacaya females serving as embryo donors were mated to males of proven fertility. Donors received 30 µg of gonadorelin at the time of breeding, and embryos were non-surgically recovered 7 days after mating. Recovered embryos (n = 4) were placed individually through a series of three vitrification solutions at 20°C (VS1: 1.4 M glycerol; VS2: 1.4 M glycerol + 3.6 M ethylene glycol; VS3: 3.4 M glycerol + 4.6 M ethylene glycol) before loading into an open-pulled straw (OPS) and plunging directly into liquid nitrogen for storage. At warming, each individual embryo was sequentially placed through warming solutions (WS1: 0.5 M galactose at 37°C; WS2: 0.25 M galactose at 20°C), and warmed embryos were incubated at 37°C in 5% CO2 in humidified air for 20-22 h in 1 ml Syngro® holding medium supplemented with 10% (v/v) alpaca serum to perform an initial in vitro assessment of post-warming viability. Embryos whose diameter increased during culture (n = 2) were transferred individually into synchronous recipients, whereas embryos that did not grow (n = 2) were transferred together into a single recipient to perform an in vivo assessment of post-warming viability. Initial pregnancy detection was performed ultrasonographically 29 days post-transfer when fetal heartbeat could be detected, and one of three recipients was pregnant (25% embryo survival rate). On November 13, 2019, the one pregnant recipient delivered what is believed to be the world's first cria produced from a vitrified-warmed alpaca embryo.

Seroprevalence and risk factors for Trypanosoma evansi, the causative agent of surra, in the dromedary camel (Camelus dromedarius) population in Southeastern Algeria.

Surra, caused by Trypanosoma evansi, is a re-emerging animal trypanosomosis, which is of special concern for camel-rearing regions of Africa and Asia. Surra decreases milk yield, lessens animal body condition score and reduces market value of exported animals resulting in substantial economic losses. A cross-sectional seroprevalence study of dromedary camels was conducted in Algeria, and major risk factors associated with infection were identified by collecting data on animal characteristics and herd management practices. The seroprevalence of T. evansi infection was determined in sera of 865 camels from 82 herds located in eastern Algeria using an antibody test (card agglutination test for Trypanosomiasis - CATT/T. evansi). Individual and herd seroprevalence were 49.5% and 73.2%, respectively, indicating substantial exposure of camels to T. evansi in the four districts studied. Five significant risk factors for T. evansi hemoparasite infection were identified: geographical area, herd size, husbandry system, accessibility to natural water sources and type of watering. There was no association between breed, sex or age with T. evansi infection. Results of this study provide baseline information that will be useful for launching control programmes in the region and potentially elsewhere.

First report of Chlamydophila abortus infection in the dromedary camel (Camelus dromedarius) population in eastern Algeria.

Chlamydiosis is caused by an obligate intracellular gram-negative bacterium of the genus Chlamydophila which is a zoonotic pathogen. The objectives of the study were to identify the seroprevalence of antibodies against Chlamydophila abortus in dromedary camel herds from four districts in eastern Algeria, as well as to estimate the association between seroprevalence and certain factors present at the animal and herd levels. Blood samples were collected from a random sample of animals within each of 82 camel herds. Serum samples were subjected to a C. abortus ELISA test, and association between the presence of antibodies and potential risk factors was estimated. Animal and herd seroprevalence were 2.5 % and 15.8 %, respectively, indicating substantial exposure of camels to C. abortus in the four districts studied. Age, breed, and sex did not influence seroprevalence in tested animals. Based on the univariate analysis, contact with sheep and goats, contact with other camel herds, and histories of abortion were major risk factors for infection. By using multivariate analysis, contact of camels with sheep and goats and with others camel herds, through shared grazing or watering points, were important factors for transmission of chlamydiosis with an odds ratio of 3.3 and 9.4, respectively. At the herd level the introduction of purchased animals was the major risk factor. This baseline information will be highly useful for launching C. abortus control programs in the region and potentially elsewhere.

Postpartum meloxicam administration alters plasma haptoglobin, polyunsaturated fatty acid, and oxylipid concentrations in postpartum ewes.

BACKGROUND: Postpartum inflammation is a natural and necessary response; however, a dysfunctional inflammatory response can be detrimental to animal productivity. The objective of this study was to determine the effects of a non-steroidal anti-inflammatory drug (meloxicam) on ewe postpartum inflammatory response, ewe plasma polyunsaturated fatty acid and oxylipid concentrations, and lamb growth. RESULTS: After lambing, 36 Hampshire and Hampshire × Suffolk ewes were sequentially assigned within type of birth to control (n = 17) or meloxicam orally administered on d 1 and 4 of lactation (MEL; 90 mg, n = 19). Milk and blood samples were collected on d 1 (prior to treatment) and d 4. Milk glucose-6-phosphate was not affected by MEL. Plasma haptoglobin (Hp) concentrations were less for MEL ewes; control ewes with greater d 1 Hp concentrations had elevated Hp on d 4, but this was not the case for MEL-treated ewes. Treatment with MEL increased plasma arachidonic acid concentration by more than 4-fold in ewes rearing singles but decreased concentrations of 9,10-dihydroxyoctadecenoic acid, prostaglandin F2α, 8-iso-prostaglandin E2, and 8,9-dihydroxyeicosatetraenoic acid. Nine oxylipids in plasma had interactions of treatment with d 1 Hp concentration, all of which revealed positive associations between d 1 Hp and d 4 oxylipid concentrations for CON, but neutral or negative relationships for MEL. MEL decreased 13-hydroxyoctadecadienoic acid:13-oxooctadecadienoic acid ratio and tended to increase 9-hydroxyoctadecadienoic acid:9-oxooctadecadienoic acid ratio (both dependent on d 1 values), indicating progressive metabolism of linoleic acid-derived oxylipids occurred by enzymatic oxidation after MEL treatment. Meloxicam reduced oxylipids generated across oxygenation pathways, potentially due to an improved redox state. CONCLUSIONS: Postpartum MEL treatment of ewes decreased plasma concentrations of Hp and several oxylipids, with the greatest impact in ewes with biomarkers reflecting a greater inflammatory state before treatment. Anti-inflammatory strategies may help resolve excessive postpartum inflammation in some dams.

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa.

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.

Seroprevalence and risk factors for Coxiella burnetii, the causative agent of Q fever in the dromedary camel (Camelus dromedarius) population in Algeria.

Query (Q) fever is a globally distributed zoonotic disease caused by Coxiella burnetii, a bacterial agent for which ruminants are the most prevalent natural reservoir. Data regarding Q fever infection in camels in Algeria are limited. Therefore, a survey to detect seroprevalence of C. burnetii antibodies was conducted among healthy camel populations in a vast area in southeastern Algeria to determine distribution of the Q fever causative organism and to identify risk factors associated with infection. Between January and March 2016, blood samples were collected from 184 camels and serum samples were subsequently analysed using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. At the time of blood collection, a questionnaire investigating 13 potential predisposing factors associated with C. burnetii seropositivity was completed for every dromedary camel and herd. Results were analysed by a chi-square (χ2) test and multivariate logistic regression. The seroprevalence of C. burnetii at the animal level was 71.2% (95% CI: 65.2-78.3) and 85.3% (95% CI: 72.8-97.8) at the herd level. At the animal level, differences in seroprevalence were observed because of herd size, animal age, animal sex, presence of ticks and contact with other herds. A multivariable logistic regression model identified three main risk factors associated with individual seropositivity: (1) age class > 11 years (OR = 8.81, 95% CI: 2.55-30.41), (2) herd size > 50 head (OR = 4.46, 95% CI: 1.01-19.59) and (3) infestation with ticks (OR 2.2; 95% CI: 1.1-4.5). This study of seroprevalence of C. burnetii infection in camels in Algeria revealed a high seroprevalence of Q fever in camel populations in southeastern Algeria and provided strong evidence that Q fever represents an economic, public health and veterinary concern. Appropriate measures should be taken to prevent the spread of C. burnetii and to reduce the risk of Q fever in farm animals and humans in this agro-ecologically and strategically important region of North Africa.

Cryopreservation of preimplantation embryos of cattle, sheep, and goats.

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 - 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.