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).

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.

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.

Positive effect of natural antioxidant oregonin from Alnus incana bark on ram semen quality stored at 5 °C for 48 h.

The maintenance of high vitality and motility of ram's spermatozoa during storage at low temperatures has a crucial role for successful fertilization. This study evaluates the effect of the natural antioxidant oregonin on ram semen quality, stored at 5 °C for 48 h. Еighteen ejaculates (three repetitions for 6 ejaculates) from three local breed rams, collecting by artificial vagina, with volume > 1 ml, concentration > 1 × 109/ml and mass motility >3.5 were used for chilling. Each ejaculate was separated in two equal parts, diluted with Tris-glucose-glycerol-egg yolk extender with no oregonin or supplemented with 100µÐœ oregonin until adjustment of the sperm concentration to 200 × 106 cells/ml and stored at 5 °C for 48 h. The semen quality assessment was based on the main kinematic (by CASA analysis), morphological parameters (by BrightVit kit staining) and mitochondrial status (by MitoView staining) of the spermatozoa on 0, 24 and 48 h of storage, and on in vivo fertility test. Oregonin did not impair the morphology and kept sustained motility of ram spermatozoa stored at 5 °C for 48 h. The curvilinear velocity indicated faster movement of the oregonin treated sperms that corresponded with high percent of spermatozoa with active mitochondria in these samples. The fertilizing capacity of spermatozoa was preserved and pregnancy rate in the experimental group was 80% versus 60% in control. In conclusion, this study provides a new data about positive effect of the natural antioxidant oregonin, supplemented to the extender, on chilled ram semen quality, including fertilizing ability.

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.

Ultrasonic evaluation of uterine involution in Bulgarian Murrah buffalo after administration of oxytocin.

The aim of this study was to determine the time taken for complete uterine involution in Bulgarian Murrah buffaloes following normal parturition and oxytocin stimulated milking; and to establish the time course of the change in size of the uterine horns, the cervix and caruncles between parturition and involution by means of ultrasonography. There were 17 animals in the study aged 3-6 years and average parity of 2.17 ± 0.18. They were administered 20 IU oxytocin 15 min before each milking. Rectal palpation and transrectal ultrasonography were performed at 3 d intervals from Days 1 to 34 post partum. The involution of the non-gravid and gravid uterine horns, and the cervix was complete by Days 22 and 25 post partum when their diameters were 2.7 ± 0.4 cm, 2.8 ± 0.3 cm and 3.12 ± 0.4 cm, respectively. Caruncles underwent rapid regression until Day 10 post partum. It was not possible to determine the dimensions of the caruncles after that time. The cumulative percentage of animals whose uterus was located in the pelvic cavity increased from 24% at Day 10 post partum to 100% at Day 34 post partum. The combination of rectal palpation and transrectal ultrasonography provided a reliable method of evaluating changes in the uterus over time and determining the time of uterine involution. The present study showed that complete uterine involution, with the uterus located in the pelvic cavity, was achieved by Day 34 after parturition in all 17 Bulgarian Murrah buffaloes treated with oxytocin before milking.