In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848.

The quality of the separated fractions in sex-sorted semen is very important for the success of the artificial insemination. This study aimed to evaluate some in vitro characteristics (DNA quantity, kinematic parameters and enzymes activity) of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and toll-like receptors (TLR)7/8 ligand R848. The ejaculates from six rams were collected by artificial vagina and subjected to a computer-assisted semen analysis (CASA). Total motility and percentage of the sperms with rapid and medium progressivity or non-progressivity in whole ejaculates and in X and Y fractions were analyzed. Activity of the enzymes ALP, GGT, CK, LDH and accumulation of lactate in the seminal plasma of ejaculates and in the environmental fluid of sexed spermatozoa were measured by biochemical analyzer. DNA was isolated from precipitated spermatozoa, and its quantity was measured. For both protocols the DNA mass from X-bearing fractions was higher, than from Y-bearing fractions. The high total motility of X- and Y-bearing spermatozoa as well as greater percent sperms with progressive motility were observed after use of BSA protocol. The application of TLR7/8 ligand R848 protocol led to reducing of Y-sperm motility and enhancement of non-progressivity in both fractions, which corresponded to the determined high amount of the extracellular lactate. For both methods, the significantly reduced activity of enzymes in the X and Y spermatozoa environmental fluids was established. Both protocols produce X- and Y-sperm fractions with satisfactory quality (over 80% total motility and over 50% rapid and medium progressive spermatozoa in each fraction).

Teat morphology across five buffalo breeds: a multi-country collaborative study.

The buffalo (Bubalus bubalis) is a species of worldwide importance, raised to produce milk, meat, and hides, and often used as a working animal in rural contexts with low access to hi-tech solutions. In the present study, 100 lactating buffaloes (50 primiparous and 50 pluriparous) of five popular breeds were recruited to characterize and compare teat morphology. In particular, the focus was put on the Nili Ravi, Mediterranean, Egyptian, Bulgarian Murrah, and Azeri buffaloes raised in Pakistan, Italy, Egypt, Bulgaria, and Iran, respectively. In all countries, a longitudinal cross-section ultrasound was obtained before the milking to measure teat parameters at individual level: overall, teat canal length (TCL) averaged 24.13 mm, teat diameter (TD) 30.46 mm, cisternal diameter (CD) 17.80 mm, and teat wall (TW) 7.12 mm. The most variable trait across breeds was TCL which was positively correlated with CD and TD and negatively with TW, regardless of the teat position (front/rear or left/right). A strong negative correlation was found between TW and CD (- 0.43). The analysis of variance revealed that the fixed effect of breed significantly affected all the traits except TD. In fact, Bulgarian Murrah, Azeri, and Egyptian buffaloes presented the greatest estimate of TCL, whereas NR the smallest (14.70 mm). The TW was maximum in Nili Ravi, Egyptian, and Mediterranean buffaloes, with estimates equal to 8.19, 7.59, and 8.74 mm, respectively. Nili Ravi also showed the greatest TL (82.39 mm). In terms of CD, the lowest least square mean was that of Mediterranean buffaloes (12.14 mm). Primiparous and pluriparous buffaloes differed in terms of TD, TW, and TL, with older animals presenting the highest least square mean. In terms of position, instead, significant differences were observed for TD, CD, and TL when comparing front and rear teats, as left and right teats did not differ. Teat anatomy includes a set of heritable morphological features and is therefore breed-dependent. Differences presented in this study could be attributed to the divergent breeding objective and selective pressure across the five breeds; e.g., in some cases such as Mediterranean buffalo, selection for decades was oriented to improve milk production and milkability and achieve optimal conformation for mechanical milking. A better understanding of the mammary gland anatomical descriptors can be informative of the history of a breed and could provide useful insights to guide possible selection.

Clinical retrospective study of vaginal hyperplasia in the bitch (2012-2022).

A retrospective study was used to analyse canine vaginal hyperplasia ambulatory cases in Small animal clinic of the University Veterinary Hospital of the Faculty of Veterinary Medicine, Trakia University in Stara Zagora, Bulgaria between 2012 and 2022. The study was done to investigate the morbidity of vaginal hyperplasia in the bitch and its relationship with the breed, body weight, age, stage of the oestrous cycle, parity of dogs and grade of the condition. Grade II and grade III vaginal hyperplasia were observed in 3.11% of all female dog genital pathological cases. Twenty-eight canine breeds were affected and the largest number were Central Asian Shepherds (30.47%). Most frequently the dogs were between 2 and 3 years old (29.68%), above 40 kg body weight (58.59%) and during follicular phase (proestrus and oestrus) of the oestrous cycle (82.02%). All the affected bitches were non-spayed (100%) and nulliparous were 70.31% of them. Grade III vaginal hyperplasia was more frequently observed (53.13%) than grade II of the condition. High correlation was found between age of the bitches and grade of the vaginal hyperplasia (R2 = .551; SEE = 0.053), body weight and grade of the condition (R2 = .820; SEE = 0.054), and age of the animals and their body weight (R2 = .820; SEE = 1.27).

Influence of Ovarian Status and Steroid Hormone Concentration on Day of Timed Artificial Insemination (TAI) on the Reproductive Performance of Dairy Cows Inseminated with Sexed Semen.

This study aimed to evaluate the effect of the ovarian status and steroid hormone concentration on the day of TAI on the reproductive performance of dairy cows subjected to estrus synchronization treatment and timed artificial insemination with sexed semen. Seventy-eight cyclic Holstein cows pre-treated with PGF2α-GnRH were divided in two groups-I (Preselect-OvSynch, n = 38) and II (OvSynch+PRID-7-day+eCG, n = 40)-and inseminated with sexed semen. The presence of preovulatory follicle (PF) with or without corpus luteum (CL), the PF diameter, the estradiol (E2) and progesterone (P4) concentrations on the day of TAI, the pregnancy rate (PR) and embryo loss were determined. On the day of TAI, 78.4% of all the pregnant cows presented a PF (mean size 1.80 ± 0.12 cm) without CL, low P4 (0.59 ± 0.28 ng/mL) and high E2 (12.35 ± 2.62 pg/mg) concentrations. The positive correlation between the size of the PF and the level of E2 in the pregnant cows from group II was stronger than that of group I (R = 0.82 vs. R = 0.52, p < 0.05). The pregnancy rate on day 30 (57.5% vs. 36.8%) and day 60 (50% vs. 26.3%; p < 0.05) and the embryo losses (13% vs. 28.5%) showed better effects of treatment in group II. In conclusion, the ovarian status and the steroid hormone concentration on the day of TAI influence the pregnancy rates of dairy cows subjected to estrus synchronization and timed artificial insemination with sexed semen.

Oxytetracycline Persistence in Uterine Secretion after Intrauterine Administration in Cows with Metritis.

Puerperal metritis in cows is often treated with antibacterial drugs. The prudent use of antibiotics in farm animals requires state-of-the-art knowledge of their pharmacokinetics and data from sensitivity tests of pathogenic bacteria. Changes in oxytetracycline levels in the uterine secretion over time after intrauterine administration in cows with metritis were evaluated in relation to the sensitivity of pathogenic bacterial isolates. Oxytetracycline levels in plasma, milk and uterine secretion were determined via LC-MS/MS analysis. Pathogenic bacteria were isolated and their sensitivity to oxytetracycline was determined. The concentrations of oxytetracycline in the uterine secretion were 433.79 (39.17-1668.76) µg·mL-1 six hours after the third application at a dose of 8 mg/kg and 84.33 (1.58-467.55) µg·mL-1 96 h after the last treatment. These levels were higher than the minimum inhibitory concentrations-namely, between 4 and 64 µg·mL-1-against pathogenic isolates Trueperella pyogenes, Streptococcus intermedius, Escherichia coli and Bacillus pumilis. Higher concentrations over time were measured in milk rather than in plasma, indicating the need to monitor the withdrawal time for milk. The intrauterine infusion of oxytetracycline for three consecutive days resulted in the successful treatment of metritis in terms alleviating inflammation and restoring the estrus cycle in cows.

The use of ultrasonographic measurement of the heart size and fetal heart rate variation for gestational age determination in local Bulgarian goats.

BACKGROUND: Determination of gestational age in goats was performed using ultrasound measurements of different fetal biometric parameters. There are no data showing quadratic and exponential dependence between fetal heart parameters and gestational age. OBJECTIVES: The objective of the present study was to test the significance of the defined indicators (fetal heart rate, longitudinal and transverse fetal heart axes) for determining gestational age in local Bulgarian goats. METHODS: A total of 24 pregnant local Bulgarian goats, aged between 2 and 5 years, body weight 42-50 kg were included in the study. Ultrasonographic examinations were performed weekly on gestational days 21, 28, 35, 42 and 49. After gestational day 49 until parturition, scans were performed biweekly. The data from the measurements were subjected to linear (y = a + bx), quadratic (y = aх2 + bх + c) and exponential (y = axn ) correlation. RESULTS: Average fetal heart rate values decreased with pregnancy progression. The coefficient of determination (R2 ) and standard error of estimate (SEE) of the heart rate indicator were 0.72, 0.75, 0.58 and 15.1, 14.4, 19.2 days, respectively, for the three correlations. Longitudinal diameter (long axis) had the following values for R2 = 0.94, 0.96, 0.96 and for SEE = 7.0, 5.5, 5.5 days, for the linear, quadratic and exponential correlations, respectively, while the values of the fetal heart transverse diameter (short axis) were higher than those of the external one (R2 = 0.95, 0.97, 0.97). Simultaneously, SEE is lower (SEE = 6.1, 4.9, 5.0 days) compared to that found for the long heart fetal axis. CONCLUSIONS: For precise estimation of gestational age, use of longitudinal (long) and transverse (short) axes is recommended. Fetal heart rate is not an exact indicator but can be used as a reference for gestational age along with changes in the heart size and echogenicity.

Doppler ultrasound measurements of the blood flow velocity in the fetal heart and aorta in Bulgarian White milk goats.

The aim of the present study was to determine the parameters characterising the blood flow velocity in the fetal heart and aorta in goats by Doppler ultrasonography. Twenty-four Bulgarian White milk goats, aged 4-6 years, weighing 45-51 kg were used in the study. Doppler ultrasound measurements of the blood flow velocity in fetal heart were done at the end of first, second and third trimesters of pregnancy, and in the fetal aorta - at the end of the second and third trimesters of pregnancy. Ultrasound investigations were carried out by transabdominal approach with linear and convex transducers, with frequency 8.0 MHz. Blood flow parameters that included maximum and minimum systolic velocity, end-diastolic velocity, pulsatility index, resistance index and systolic/diastolic ratio were measured by spectral Doppler ultrasonography. The results showed that the highest maximum systolic velocity in the fetal heart was observed at the end of the first pregnancy trimester. Statistically significantly (p < 0.05) lower values of this parameter were registered in the second and third pregnancy trimesters. A similar tendency was observed for minimum systolic velocity, end-diastolic velocity and systolic/diastolic ratio. The pulsatility index and resistance index had similar values for the three studied periods. Examination of the fetal aorta showed statistically significant differences (p < 0.05) of minimum systolic velocity, end-diastolic velocity, pulsatility and resistance index in the third, compared with the second trimester of pregnancy. The analysis of the results suggested that Doppler ultrasonography can be used to monitor fetal blood flow changes during the various stages of pregnancy.

Resumption of Cyclic Ovarian Activity by Herbal Preparation AyuFertin in Bulgarian Murrah Buffaloes at Early Postpartum.

This study evaluates the phytochemical composition and ability of herbal preparation AyuFertin, supplemented shortly after calving, to resume the cyclic ovarian activity in postpartum Bulgarian Murrah buffaloes. In total, 13 healthy Bulgarian Murrah buffaloes at the age of 4-10 years, calved in March-April 2019 were involved in the experiment. Seven experimental buffaloes were treated individually per os with AyuFertin according to producer instruction. All animals were subjected to regular ultrasound examination of ovaries. GC-MS analysis of fatty acids composition and HPLC-DAD analysis of carotenoid and tocopherol content in AyuFertin were conducted. The determination of estrus occurrence and natural mating were done by a fertile bull. The development of the large follicles (12.5-14.3 mm) in 85.7% of treated animals was observed on day 24 postpartum compared to 0% in controls. Clinical signs of estrus were recorded in 71.4% of the animals, followed by an 80% pregnancy rate versus 16% in controls within 70 days after calving. In conclusion, the supplementation of the bioactive herbal product AyuFertin from day 21 postpartum can stimulate the follicular growth in the buffalo's ovary, which leads to the development of follicles with ovulatory capacity, followed by successful ovulation. The above-mentioned treatment resulted in a better pregnancy rate in the treated multiparous compared to the control buffaloes bred by natural service.

Oxytetracycline Pharmacokinetics After Intramuscular Administration in Cows with Clinical Metritis Associated with Trueperella Pyogenes Infection.

Systemic therapy with oxytetracycline is often used for treatment of clinical metritis although data about its penetration into the uterus and uterine secretion are lacking. Uterine secretions and milk from six cows with clinical metritis were collected for microbiological assay. The animals were treated intramuscularly with long-acting oxytetracycline (20 mg/kg) and samples of plasma, milk and uterine secretions were collected for determination of the antibiotic concentrations by HPLC-PDA analysis. Pharmacokinetics of the antibiotic and in silico prediction of its penetration into the uterus were described. Trueperella pyogenes with MIC values of 16-64 µg mL-1 was isolated (n of cows = 4) from uterine secretions. Oxytetracycline showed fast absorption and penetration in the uterine secretions and milk. No change of withdrawal time for milk was necessitated in cows with clinical metritis. Maximum levels in uterine secretions and predicted concentrations of oxytetracycline in the uterus were lower than MIC values. Systemic administration of long-acting oxytetracycline did not guarantee clinical cure and was not a suitable choice for treatment of clinical metritis associated with Trueperella pyogenes. The appropriate approach to antibiotic treatment of uterine infections of cows requires knowledge on penetration of the antibiotics at the site of infection and sensitivity of pathogens.

Determination of the gestational age through ultrasound measurements of some uterine and foetal parameters in Bulgarian local goats.

The aim of the current study was to determine the gestational age in goats through the measurement of some foetal and uterine structures. Twenty-four pregnant Bulgarian local goats were submitted to ultrasound foetometry by transrectal and transabdominal approaches. Ultrasound measurement of studied parameters was done at 7-day intervals during the first trimester of gestation (days 21-49) and at 14-day intervals between gestation days 49 and 133. Associations of foetometry data with foetal age were investigated by linear (y = a + bx), quadratic (y = ax2  + bx + c) and exponential (y = axn ) equations. According to the results, some biometric parameters (uterine lumen diameter, crown-rump length, trunk diameter, biparietal diameter, foetal aortic diameter) exhibited a stronger correlation to gestational age (R2  ≥ 0.90) and lower standard error of the estimate (SEE ≤ 8) as compared to foetal eye orbit diameter, and outer and inner placentome diameters (R2  ≤ 0.90; SEE ≥ 8). Each of these biometric parameters could be measured in different periods of gestation, thus allowing for monitoring and determination of foetal age at almost all stages of pregnancy.

Basal testosterone concentrations after the application of a slow-release GnRH agonist implant are associated with a loss of response to buserelin, a short-term GnRH agonist, in the tom cat.

Slow-release GnRH agonist implants are considered an effective, reversible alternative to surgical castration in male tom cats. Individual differences exist regarding the onset of efficacy and might be delayed in some animals. Single measurements of testosterone (T) might result in basal concentrations also in intact male cats. Consequently, GnRH stimulation tests are performed to measure T increase in intact animals and to differentiate castrated from intact male cats. In this study, five tom cats were treated with a 4.7-mg deslorelin implant and GnRH stimulation tests using buserelin were performed before treatment and at 4-week intervals afterward until Week 20. After the last test in Week 20 all animals were castrated. Four of five animals had basal T after 4 weeks and-in contrast to pretreatment-application of buserelin did not result in any further T increase. In one animal, T was low after implant insertion, but not basal; however, a GnRH stimulation test induced a slight increase of T in Week 8 and 16 only and no response in Weeks 4, 12, and 20. Testicular volume was significantly decreased and penile spines disappeared in all cats. Testicular histology showed mixed atrophy, but also fully elongated spermatids in three of five male cats making infertility questionable. Because of the loss of the stimulatory effect of short-term GnRH application (buserelin), it can be assumed that long-term GnRH agonists also act by some mechanisms of downregulation of pituitary GnRH receptors in the tom cat.