Regenerative Potential of Mesenchymal Stem Cells for Enhancing Uterine Health and Fertility in Repeat Breeder Dairy Cows Bruno Leonardo Mendonça Ribeiro, Joice Fülber, Mario Augusto Reyes Aleman, Luiz Francisco Machado Pfeifer, Jéssica de Souza Andrade, et al. Stem Cells International, 2026 The suboptimal reproductive performance of repeat‐breeding (RB) cows is a major challenge for the dairy industry, leading to higher costs, prolonged calving intervals, and reduced productivity, negatively impacting herd productivity and economic viability. Among the associated factors, endometrial degeneration stands out, characterized by the replacement of functional tissue with fibrotic tissue, compromising uterine receptivity. In this context, regenerative therapy using mesenchymal stem cells (MSCs) has emerged as a promising alternative. This study evaluated the effects of intrauterine MSC inoculation in RB cows diagnosed with endometrial degeneration. Nine crossbred cows (Gyr × Holstein) were included and underwent clinical, cytological, microbiological, histopathological, ultrasonographic, and molecular evaluations on day 0 (pre‐treatment) and day 30 (post‐treatment). The results demonstrated endometrial remodeling, with fibrotic tissue replaced by loose connective tissue, increased vascularization, and the presence of new groups of endometrial glands. Doppler ultrasonography revealed enhanced blood flow of the endometrial mucosa and thickening of the uterine wall after therapy. qRT‐PCR analysis indicated reduced expression of pro‐inflammatory cytokines (IL‐1 β and IL‐8), suggesting modulation of the uterine environment. Despite the observed tissue improvement and absence of adverse effects on ovarian function, none of the inseminated cows conceived. In conclusion, MSC therapy promoted favorable changes in the endometrium and uterine environment, although it did not result in pregnancy, highlighting the need for further studies to optimize dosage, administration route, and therapeutic response time.
Effects of sperm cryopreservation extenders and estrus in pregnancy of Bos indicus cows submitted to TAI Luiz Francisco Machado Pfeifer, José Roberto Potiens, Jessica de Souza Andrade, Eriklis Nogueira Semina Ciencias Agrarias, 2025 The objective of this study was to evaluate the relationship between estrus behavior of cows and the semen’s cryopreservation extenders on the fertility of B. indicus cows subjected to timed artificial insemination (TAI). In this study, 505 postpartum B. indicus Nelore beef cows were enrolled in a TAI program and separated into groups according to the estrus detection on the Day of the TAI procedure, into: Cows detected in estrus (E, n = 307), and cows not detected in estrus (NE, n = 198). The semen used in this study was cryopreserved with 2 different extenders: Optixcell (OPT), and the egg yolk-Tris-glycerol (EYTG), and were distributed to inseminate E and NE cows. The pregnancy per AI (P/AI) of the groups EOPT (69.5 %), and EEYTG (60.1 %) were greater (P ≤ 0.05) in comparison with groups NEOPT (46.0 %), and NEEYTG (42.9 %). The group EOPT tended (P = 0.08) to have greater P/AI in comparison with the group EEYTG. In conclusion, among cows exhibiting estrus, those inseminated with semen diluted in OPT showed a tendency toward higher pregnancy per AI compared to those inseminated with EYTG-diluted semen.
Luteal Function and Pregnancy Rate in Bos taurus Heifers Treated With GnRH Prior to Timed Artificial Insemination Julia Nobre Blank Camozzato, Bruna Favretto de Souza, João Marcello Rodrigues, Alinne Machado Petrarca Léo, Luiz Francisco Machado Pfeifer, et al. Reproduction in Domestic Animals, 2025 The study aimed to evaluate the effects of GnRH given 34 h after progesterone (P4) intravaginal device (IVD) removal in a timed artificial insemination (TAI) protocol. In Experiment 1, 17 Hereford and Braford heifers (control, n = 7; GnRH34; n = 10) received 2 mg estradiol benzoate (EB) and a 1 g P4 IVD on Day 0 (D0). On D8, IVDs were removed and 150 μg d‐cloprostenol (PGF) and 1 mg estradiol cypionate (EC) were administered. In the GnRH34 group, animals received 25 μg lecirelin 34 h after the IVD removal (D9). TAI was performed in both groups on D10 (48 h after IVD removal). Follicular dynamics were evaluated from D8 until ovulation; blood samples were collected 7 and 12 days after TAI. In Experiment 2, to evaluate the effect of GnRH34 on pregnancy rates (control, n = 187; GnRH34; n = 203), heifers were subjected to the TAI protocols described in Experiment 1. No significant differences were observed in preovulatory follicle (POF) diameter on D10, follicular growth and ovulation rates until 72 h and P4 concentrations on days 7 and 12 after TAI (p > 0.05). Pregnancy rates (PR) were similar between groups (Control = 50.2%; GnRH34 = 50.2%; p = 0.65). A significant effect of body weight (BW) and body condition score (BCS) on PR was observed for control (p< 0.05), but not for GnRH34. In conclusion, GnRH 34 h after the IVD removal in beef heifers previously treated with EC had no effect on luteal function and fertility.
Effect of the Application of PGF2α Associated With Ovulation Induction in a Fixed-Time Superovulation Programme for Precocious Nellore Heifers Tamires Korchovei Sanches, Pamela Mara Celestino Soares, Marcio de Oliveira Marques, Rubens Cesar Pinto da Silva, Luiz Francisco Machado Pfeifer, et al. Reproduction in Domestic Animals, 2024 This study evaluated the effect of prostaglandin F2α (PGF2α) associated with gonadotropin‐releasing hormone (GnRH) for ovulation induction in precocious indicus heifers submitted to a fixed‐time superovulation (SOV) programme. Precocious Nellore heifers (n = 35), aged 13 months, were subjected to the SOV protocol. On day 0 (D0), all animals received intravaginal insertion of a progesterone (P4) device along with intramuscular administration of 2 mg of oestradiol benzoate, plus 200 IU of follicle‐stimulating hormone in decreasing doses, with 12‐h intervals between D4 and D7, in addition to 150 μg of D‐cloprostenol on D6 and device removal on D7. On D8, the donors received 10.5 μg of buserelin acetate and the treatment group received 300 μg of D‐cloprostenol/PGF2α. Artificial insemination was performed 12 h and 24 h after GnRH administration using frozen semen. On D15 of the protocol (i.e., D7 after insemination), the embryos were collected and evaluated. All animals passed through the control and treatment groups. Results were evaluated by analysis of variance using an adjusted mixed‐effects model (p < 0.05). There was no difference in the total number of embryos between the control and treatment groups (10.40 ± 1.52 vs. 9.60 ± 1.36; p = 0.63) or viable embryos (6.30 ± 1.22 vs. 4.30 ± 0.71). For precocious indicus heifers, treatment with PGF2α in association with GnRH did not affect embryo production in the fixed‐time SOV protocol.
Optimisation of Early Pregnancy Diagnosis and Resynchronisation in Beef Cows Audrey Bagon, Luiz Gustavo Bruno Siqueira, João Henrique Moreira Viana, Luiz Francisco Machado Pfeifer Reproduction in Domestic Animals, 2024 The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler‐based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol‐progesterone–based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1–12.9 mm (n = 64), 13.0–14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 μg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF‐based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.
Effect of maternal age on ATP content and distribution of mitochondria in bovine oocytes Dinesh Dadarwal, Luiz Pfeifer, Miriam Cervantes, Gregg P. Adams, Jaswant Singh Plos One, 2024 Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18–20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows.
Does GnRH treatment at TAI regardless of estrus occurrence increase pregnancy rate in crossbred Bos taurus suckled cows? Vanessa Silva Fernandes, Gabriella dos Santos Velho, Mateus Felipe Osório dos Santos, Kelly Alves Evangelista, Bernardo Garziera Gasperin, et al. Animal Reproduction, 2024 The impact of GnRH treatment on the day of TAI in beef cows has received limited investigation, especially concerning its association with estrus expression. Consequently, two experiments were conducted to assess the potential of GnRH treatment on the day of TAI to enhance fertility according to the expression or not of estrus in beef cows. Experiment 1 aimed to determine ovulation rate and luteal function, while Experiment 2 aimed to determine the effect of the two GnRH treatment approaches on pregnancy rate. In Experiment 1, multiparous Brangus suckling cows (n = 17) were submitted to an 8-day TAI protocol. Estrus occurrence was evaluated based on chalk removal on D10 (TAI) and cows were assigned to receive GnRH (25µg lecirelin; im) according to the group: GnRH (n = 7), regardless of estrus expression; or selectGnRH (n = 10), only cows not detected in estrus. Ovulation rate occurring until 77h after IVD removal did not differ (p = 0.17) between GnRH (85.7%; 6/7) and selectGnRH (100%; 10/10). Also, corpus luteum size and serum progesterone concentration were not affected (p>0.05) by treatments. In Experiment 2, crossbred taurine suckled cows (n = 384) were submitted to the same protocol as described in Experiment 1 and were randomly allocated to GnRH or selectGnRH groups. There was no difference in P/AI between groups (selectGnRH = 55.6%; GnRH = 54.3%; p = 0.7) 30 days after TAI. As expected, there was a pronounced effect (p<0.0001) of estrus expression on P/AI (Estrus = 61.5%; No estrus = 33.0%), regardless of group. In summary, ovulation timing and rate and luteal function did not differ between groups. Also, GnRH administration only in cows that do not show estrus is recommended, considering hormone savings and similar conception rate.
Prostaglandin e2 induces ovulation in prepubertal mice Jéssica de Souza Andrade, Juliana Pavan Zuliani, Jaswant Singh, Sulamita da Silva Setúbal, Renata Reis da Silva, et al. Brazilian Journal of Veterinary Research and Animal Science, 2021
