E2 to P4 ratio is associated with conceptus attachment in dairy cows receiving AI after double-Ovsynch but not estrus.

Prediction of pregnancy survival in lactating dairy cows can be determined by the conceptus attachment timeframe via daily pregnancy-specific protein B (PSPB) monitoring. All factors contributing to reduced fertility in dairy cows receiving AI following estrus detection remain unclear. This study aimed to determine differences in time to conceptus attachment in lactating cows treated with the fertility program Double-Ovsynch compared to cows that were detected in estrus. Additionally, we investigated various pre- and post-conception factors potentially influencing fertility outcomes. We hypothesized that AI following a natural estrus detected with automated activity monitors would lead to an extended time to conceptus attachment and lower PSPB concentrations post-attachment compared to Double-Ovsynch. There were no differences in the average time to conceptus attachments between treatments. However, cows inseminated post-estrus that experienced pregnancy loss between conceptus attachment and 60-66 days post-AI exhibited diminished PSPB concentrations on days 2 and 3 following conceptus attachment. Steroid hormone interactions were assessed with radioimmunoassay to determine the ratios of estrogen to progesterone concentrations on the day of the luteinizing hormone (LH) surge. Notably, estrogen to progesterone ratio proved to predict conceptus attachment in cows subjected to Double-Ovsynch but not in those inseminated post-estrus detection surge. In conclusion, the estrogen to progesterone ratio measured around the time of the pre-ovulatory LH surge emerges as a potentially effective tool for estimating the fertility potential of lactating dairy cows undergoing timed AI, particularly in the context of the Double-Ovsynch program.

Reduced period from follicular wave emergence to luteolysis generated greater steroidogenic follicles and estrus intensity in dairy cows.

The onset of productive life in dairy cattle, concomitant to parturition, is accompanied by a substantial decrease in fertility in comparison with non-lactating, nulliparous heifers. Follicular growth patterns differ between parous and nulliparous dairy cattle. Nulliparous heifers ovulate follicles with reduced antral age (RAA). This study aimed to exogenously reduce ovulatory follicle age in lactating dairy cows from 7 to 5 days old. Cows (n = 80) had their estrous cycles synchronized with the Double-Ovsynch program. At the final portion of this program, luteolysis was induced at either 5 (RAA) or 7 (Control) days following follicular wave emergence. RAA outcomes were estimated in comparison with Controls. RAA resulted in smaller follicles 2 days post-treatment. Despite lower serum concentrations of 17ß-estradiol before treatment compared with Controls, the rate of increase in this hormone was greater for the RAA treatment. There was no difference in luteolysis rates between treatments. Proestrus (luteolysis onset to estrus onset) was prolonged in RAA cows. Cows with RAA had more intense estruses. Collectively, these results indicate that decreasing the age of the ovulatory follicle may improve the steroidogenic capacity of the dominant follicle and estrus expression intensity in lactating dairy cows.

Time to increase in pregnancy-specific protein B following artificial insemination is a direct determinant of subsequent pregnancy loss in lactating dairy cows.

Increasing progesterone (P4) during early conceptus development may be crucial for establishment of pregnancy in dairy cattle. The objective of this study was to determine if human chorionic gonadotropin (hCG) at various times after ovulation will increase serum P4 during elongation and increase the chances for, and reduce variability to, initial increase in pregnancy-specific protein B (PSPB) following artificial insemination (AI). Time to PSPB increase was defined as the first day of increase in concentrations of PSPB between d 18 and 28 after ovulation in cows with ≥12.5% increases for 3 consecutive days compared with baseline. Lactating cows (n = 368) synchronized to Double-Ovsynch (first service) or Ovsynch (second or greater service) received one of 4 treatments: no hCG (control), or 3,000 IU of hCG on d 2 (D2), 2 and 5 (D2+5), or 5 (D5) after ovulation. All cows were examined via ultrasound on d 5 and 10 postovulation to determine percentage of cows with hCG-induced accessory CL (aCL) and to quantify and measure all luteal structures. Samples for serum P4 were collected on d 0, 5, 19, and 20 postovulation. The P4 was increased in D2, D2+5, and D5 groups compared with control. The D2+5 and D5 treatments increased aCL and P4 compared with D2 and control. The D2 treatment increased P4 on d 5 after ovulation compared with control. Serum PSPB samples were collected daily from all cows on d 18 through 28 after ovulation for determination of d of PSPB increase. Pregnancy diagnoses were performed via ultrasound examination on d 35, 63, and 100 after ovulation and AI. The D5 treatment reduced percentage of cows with, and increased the time to, PSPB increase. Primiparous cows with ipsilateral aCL had reduced pregnancy loss before d 100 postovulation compared with cows with contralateral aCL. Cows that had PSPB increase >21 d postovulation had 4× greater chances of pregnancy loss compared with cows that had PSPB increase on d 20 or 21. The highest quartile of P4 on d 5, but not on d 19 and 20, was associated with reduced time to PSPB increase. Time to PSPB increase appears to be an important measurement to understand reasons for pregnancy loss in lactating dairy cows. Increasing P4 utilizing hCG after ovulation did not enhance early pregnancy or reduce pregnancy losses in lactating dairy cows.

Dairy heifers have an earlier increase in serum pregnancy-specific protein B compared with lactating dairy cows. Is this an indicator of earlier conceptus attachment?

This study was designed to use within-individual daily increases in circulating pregnancy-specific protein B (PSPB) to determine time to increase in serum PSPB in nulliparous heifers and multiparous dairy cows following artificial insemination (AI). Weekly cohorts of lactating Holstein cows (n = 56) averaging 122 ± 7 d in milk at AI and nulliparous Holstein heifers (n = 23) averaging 16 ± 0.24 mo old at AI were used in this study. Lactating cows received AI to either Ovsynch (n = 38) or at ~12 h following observed standing estrus (n = 18). All heifers were inseminated ~12 h after observed standing estrus. Blood samples for measurement of PSPB were collected daily from d 15 through d 35 postovulation. Nulliparous heifers had increases in serum PSPB earlier compared with both primiparous and multiparous cows. Day of increase in serum PSPB was defined as the day serum PSPB optical density levels initially increased ≥10% from baseline and continued to increase from baseline of ≥10% the following 2 d. Average PSPB were greater in pregnant heifers compared pregnant cows from d 23 through 29. Early lactation nonpregnant cows maintained greater average optical density of serum PSPB from 15 to 35 d postovulation compared with nonpregnant heifers (n = 38). In summary, fertility differences in heifers versus lactating cows may be due to the differences in timing of increases in serum PSPB. This appears to be one of the first publications that used daily PSPB sampling to investigate possible differences in fertility in heifers versus lactating cows.

The effect of a double dose of cloprostenol sodium on luteal blood flow and pregnancy rates per artificial insemination in lactating dairy cows.

Inadequate luteolysis in fertility programs is a problem for lactating dairy cows treated with a single dose of PGF2α. The proportion of cows with complete luteolysis can be increased by administering 2 doses of PGF2α 24 h apart. This study hypothesized that a double dose of cloprostenol sodium (1.0 mg) could take the place of 2 doses 24 h apart due to its enhanced half-life. Cows were allocated to receive 1 of 3 treatments: negative controls: 0.5 mg of cloprostenol sodium (single; n = 337); positive controls: two 0.5-mg doses of cloprostenol sodium 24 h apart (two/24; n = 313); and treated: 1.0 mg of cloprostenol sodium (double; n = 298) at the final PGF2α of Double-Ovsynch. Cows received artificial insemination (AI) 16 h after final GnRH of Double-Ovsynch. Pregnancy diagnosis was determined at 24, 34, 62, and 184 d post-AI. Pregnancy loss was categorized in the following periods: between 24 to 34, 34 to 62, and 62 to 184 d post-AI. Ultrasonography (B-mode and color Doppler) was used to assess luteal function pre- and posttreatment with various doses of cloprostenol sodium. Luteal volume and luteal blood flow (LBF) from d 7 and 14 corpora lutea were determined before treatment (d -1), and 2 and 4 d after treatment. No evidence was observed of an effect of treatment on pregnancy rates per AI at 24, 34, or 62 d post-AI. No effect was observed of treatment on pregnancy losses occurring between d 24 and 34, 34, and 62, and between 62 and 184 d post-AI. However, third-plus parity cows treated with the single treatment had greater pregnancy loss compared with two/24 and double between d 24 and 34 post-AI. Third-plus parity cows that received the double treatment had lower LBF 2 and 4 d after treatment compared with cows treated with single. Amount of LBF present 4 d after treatment was not a predictor of pregnancy or pregnancy loss. A double dose (1.0 mg) of cloprostenol sodium may be a feasible alternative for fertility programs based on nondifferent outcomes to the two/24 treatment, lower pregnancy losses, and reduced LBF disappearance following treatment in third-plus parity cows in comparison with the single treatment.

Effect of cloprostenol sodium dose on luteal blood flow and volume measurements in Holstein heifers with both day-4 and day-10 corpora lutea.

Establishment of a vascular system within the corpus luteum (CL) is critical for progesterone (P4) secretion. Measurement of luteal blood flow (LBF) may be a feasible way to determine luteolysis induced with cloprostenol sodium (CLO). Our overall objective was to establish timelines to assess luteolysis via Doppler ultrasonography. Estrous cycles were synchronized in 11- to 12-mo-old Holstein heifers (n = 37). Heifers were injected CLO at a random stage of the estrous cycle 12 d from treatment. Gonadotropin-releasing hormone (GnRH) was administered 2 (d -10) and 8 d (d -4) after the initial CLO. This satisfied the study objective of inducing simultaneous presence of CL at d 4 of development (D4 CL) and CL at d 10 of development (D10 CL) on the day of treatment with different CLO doses (hereafter referred to as d 0). Heifers were randomly assigned to 1 of 5 treatments on d 0: negative control (NC) consisting of no treatment with CLO (n = 8); a quarter dose of CLO (0.125 mg; n = 8); half dose of CLO (0.25 mg; n = 8); full dose of CLO (0.5 mg; n = 8); or positive control (PC) consisting of 4 doses of 0.5 mg of CLO at 24-h intervals starting at d 0 (n = 5). Data collection was performed at d 0 (before and 1 h after treatment) and 2, 4, 6, and 8 d following treatment, to determine luteal volume (LV), LBF, and circulating concentrations of P4. Both NC and PC were efficient in mimicking physiological scenarios that occur during normal luteal development and luteolysis. Heifers that received PC had complete LBF disappearance of both D4 and D10 CL between d 2 and 4 after the first of 4 CLO treatments given 24 h apart (average 4.0 ± 0.0 and 3.2 ± 0.7 d, respectively). Complete LBF disappearance was used as a luteolysis marker. Treatment with different doses of CLO did not impair luteal development of the D4 CL. However, concurrent complete LBF disappearance for D10 CL in heifers treated with half (5/8 heifers) and full doses of CLO (8/8 heifers) resulted in less LBF in the half dose, and less LV and LBF in the full-dose treatment, in D4 CL at d 8 post-treatment, compared with NC. Treatment with various doses of CLO induced an acute increase in LBF 1 h after treatment, regardless of dose in D10 but not in D4 CL. We found a lack of dose response in LV reduction of D4 and D10 CL. Interestingly, LV of the D10 CL decreased in untreated NC between d 0 and 8 after treatment (d 10-18 of luteal development). Assessment with color Doppler ultrasound was sensitive enough to identify dose-response patterns in Holstein heifers (absence, partial, or complete luteolysis) following various doses of CLO. Variability in time to complete LBF disappearance of mature D10 CL following a full dose of CLO limits the use of Doppler ultrasonography to detect luteolysis at a single time point following treatment.

Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C / [Efeito das técnicas de separação de plasma e de seleção de esperma na recuperação e viabilidade do sêmen de garanhão pônei resfriado por 48h a 5°C]

Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 106 sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.(AU)

As técnicas de separação do plasma seminal (centrifugação, SpermFilter) e de seleção espermática (Androcoll-E e filtração por lã de vidro) foram aplicadas em 24 ejaculados de seis garanhões da raça Pônei Brasileiro. Após coleta e separação da fração gel, os ejaculados foram diluídos 1:1 com diluente à base de leite em pó. No experimento 1, os ejaculados foram distribuídos em controle (sem centrifugação), centrifugação a 600g x 10min, SpermFilter e filtração por lã de vidro. No experimento 2, o sêmen foi submetido aos procedimentos: centrifugado (SC), centrifugado com Androcoll-E e filtrado por lã de vidro. Após os procedimentos de ambos os experimentos, as amostras foram mantidas refrigeradas a 5ºC, com 50 x 106 espermatozoides/mL, por 48h. As variáveis mensuradas a fresco, 24h e 48h foram: pH, motilidade, funcionalidade de membrana, viabilidade por diacetato de carboxifluoresceína e iodeto de propídio (CFDA/PI, vitalidade (eosina/nigrosina) e atividade mitocondrial. Já osmolaridade e morfologia espermática foram avaliadas somente imediatamente após a coleta. No experimento 1, a centrifugação para retirada do plasma seminal resultou em maiores danos aos espermatozoides do que a separação por SpermFilter. A filtração por lã de vidro mostrou-se mais eficiente em separar células viáveis e manter a viabilidade durante o resfriamento. No experimento 2, os tratamentos com Androcoll-E e filtrado por lã de vidro foram superiores (P<0,0001) ao sêmen centrifugado quanto à motilidade, à funcionalidade de membrana, à atividade mitocondrial e à viabilidade, tanto nas amostras de sêmen fresco como de sêmen refrigerado. O Androcoll-E e a lã de vidro permitiram manter por 48h, a 5ºC, o sêmen de garanhões pôneis utilizando-se diluente à base de leite.(AU)

The high-fertility cycle: How timely pregnancies in one lactation may lead to less body condition loss, fewer health issues, greater fertility, and reduced early pregnancy losses in the next lactation.

Body condition loss during early lactation varies among cows in the herd and is associated with future health and reproductive outcomes. The objective of this study was to gain a greater understanding of the relationship between previous calving interval and body condition change during the first 30 d in milk (DIM) and their relationship to subsequent fertility and health variables and sex ratio at birth. Dry cows and heifers (n = 851) from a single farm entered the study approximately 25 d before the due date. They were evaluated and given a body condition score on a 1-to-5 scale with 0.1-point increments weekly until parturition. Body condition score was assessed within 1 wk of parturition and then again 27 to 33 DIM. Previous calving interval, gestation length, periparturient health events (giving birth to twins, dystocia, retained placentas, ketosis, metritis, and displaced abomasum), sire net merit ($), and milk data were used for each cow as recorded in PCDART (Dairy Records Management Systems, Raleigh, NC) by the herd managers. Longer previous calving intervals were related to greater body condition at parturition and body condition loss during the first 30 DIM. A 75% greater proportion of cows with a calving-to-pregnancy interval shorter than 130 d maintained or gained body condition during the first 30 DIM compared with cows with calving-to-pregnancy intervals longer than 130 d. Multiparous cows that maintained or gained body condition (n = 144) had greater pregnancies per AI following first service compared with cows that lost body condition (n = 577) during the first 30 DIM when health events were considered or removed. When cows with health events were considered, multiparous cows that maintained or gained body condition had a greater percentage pregnant by 130 DIM compared with cows that lost body condition (67 vs. 55%; n = 522). Cows that lost body condition during the first 30 DIM regardless of health events experienced greater pregnancy loss (n = 224) between 35 and 60 d after first AI (0.0 vs. 6.7%) compared with cows that maintained or gained body condition (n = 69) during that period. Based on data in this study from a single herd, maintaining a cycle of pregnancy before 130 DIM may reduce the amount of body condition lost after the next parturition, enhance subsequent pregnancies per AI, and reduce the possibility of early pregnancy loss. We refer to this phenomenon as the high-fertility cycle.

Response to cooling of pony stallion semen selected by glass wool filtration.

The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Retained semen was considered the portion of cells that did not transpose glass wool barrier. Total motility from the control, filtered and retained groups after 24 h of cooling was 35.5%, 43.3% and 10% (p < .0001) respectively. Sperm membrane integrity percentage at the CFDA/PI test was 37.9%, 44.8% and 14.8% (p < .0001), on the control, filtered and retained groups respectively. The results confirmed that the passage of spermatozoa through glass wool increased the selection of spermatozoa from pony stallions with higher motility, mitochondrial viability and membrane integrity for cooling in milk extender up to 24 h. Moreover, it was not obtained higher sperm parameters to control after cooling 48 h under the conditions that the study was conducted.