Natural Breeding Season Research Articles

PSXII-11 Impact of pre-breeding and breeding supplementation in May-born range heifers on growth rates and reproductive performance

Abstract Our objectives were to determine the impact rumen undegradable protein (RUP) supplementation prior to and during the breeding season had on growth rates and reproductive performance in May-born yearling range heifers. This study was conducted over a 3-yr period at the University of Nebraska, Gudmundsen Sandhills Laboratory (GSL) near Whitman, NE and utilized May-born Red Angus x Simmental crossbred heifers [n = 192; weaning body weight (BW) = 200 kg ± 28 kg]. Heifers were randomly assigned by pre-breeding BW to one of two treatments: 1) received 0.908 kg of a dried distillers grain supplement (DDGS; Supp, n = 66) pre-breeding through the breeding season or 2) received no supplement (NoSupp, n = 138). Each year, treatments were initiated in July and ended in August (55 ± 3 d). Using a C-Lock Smart Feeder (Rapid City, SD) heifers had individual access to their allotted DDGS supplementation. On average, NoSupp heifers consumed 0.09 kg/d (yr 1) and 0.02 kg/d (yr 2 and 3); whereas, Supp heifers averaged 0.59, 0.74, and 0.76 kg/d in yr 1, 2, and 3, respectively. Heifer BW was collected pre-trial (May), pre-breeding (July), breeding (August), pregnancy diagnosis (October), and pre-calving (April). Body condition score (BCS) was collected at pregnancy diagnosis (October) and pre-calving (April; Yr 1 and 2). Before each breeding season, two blood samples were collected 10 d apart to determine pubertal status. Heifers were synchronized with a single 5 mL intramuscular injection of prostaglandin F2 alpha (Lutalyse, Zoetis, Parsippany, NJ) 5 d after bull turnout during a 30 d natural service breeding season. Pregnancy diagnosis was determined via rectal ultrasound in October. No differences (P > 0.12) in BW were noted among treatments for all measurement timepoints. Moreover, SUPP heifers had greater (P < 0.02) average daily gain (ADG) from pre- trial to pregnancy diagnosis compared with NoSUPP heifers. However, there was no differences (P = 0.11) in ADG from pregnancy diagnosis to pre- calving. At pregnancy diagnosis (October), a treatment x year interaction (P = 0.02) for BCS was present where Supp heifers had a greater BCS than NoSupp heifers in yr 3. Heifer BCS pre-calving was not affected (P = 0.12) by treatment. There was a greater percentage (P < 0.01) of SUPP heifers cycling compared with NoSUPP heifers. However, there were no differences (P > 0.24) in pregnancy rates, the percentage that calved in the first 21 d, calf birth BW, or subsequent calving date. Ultimately, this study implies that providing a RUP supplement prior to and during the breeding season will increase the percentage of heifers that are cycling before breeding and have a greater ADG through pregnancy diagnosis; however, supplementation will not impact other reproductive parameters or BW gains.

PSIX-20 Differential Gene Expression in Peripheral White Blood Cells of Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef heifers remains a major economic loss to the cow-calf production industry. Identifying beef heifers with superior genetic potential for improved fertility would increase profitability. Therefore, the study aimed to identify differences in transcriptome profile from peripheral white blood cells (pWBCs) of beef heifers with varying reproductive potential. To accomplish this, Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-Synch + CIDR). Fourteen days after AI, all heifers were exposed to fertile bulls for a 60-day natural breeding season. Depending on the presence or absence of conceptus 120 days post-AI, heifers were classified as fertile (pregnant heifers) or sub-fertile (non-pregnant by AI or bull-breeding). Pregnancies were terminated, and all animals in both groups were cycling when pWBCs were isolated. Total RNA was extracted from the PWBCs and subjected to the library preparation and sequencing on the Nova-Seq platform. The read counts were obtained after data filtering and quality control using FastQC v0.11.9 and MultiQC v1.12 and alignment to the Ensemble’s ARS UCD1.2 Bos taurus genome reference using STAR aligner v2.7.5 for fertile (n = 8) and sub-fertile (n = 5) beef heifers. The filtered data were subjected to differential expression analysis using DESeq2. We identified 300 significantly differentially expressed genes (DEGs) with P-value ≤ 0.05 and absolute (log2 fold change) ≥ 0.5. Among them, 34 genes including FKBP5, ADCY3, REPS1, LAMP1, CTSH, ADGRE5 were identified with padj < 0.05. DEGs were over-represented for pathways such as response to thyroid stimulus, immune response, response to lipid, neutrophil degranulation, and calcium ion binding. Some of these genes were reported as fertility-related, while the other genes are novel and have the potential to predict heifers with varying reproductive potential. A detailed understanding of the underlying biological mechanisms of the top genes and a follow-up study with a higher sample size at different time points could validate the potential candidates identified in this study.

Influence of Hormonal Stimulation on the Oocyte Chromosome Apparatus of the Common Frog

Late vitellogenic oocytes of the common frog, Rana temporaria, represent a promising model for studying the behavior of meiotic chromosomes, since at the diplotene stage, they unite into a karyosphere, which in R. temporaria is believed to have an extrachromosomal capsule – unlike in Xenopus laevis, a classic model object of cell biology and developmental biology. However, in comparison with Xenopus, the strict breeding seasonality of R. temporaria significantly limits the possibility of using its oocytes as an experimental model. By adapting classical hormonal stimulation protocols proposed for anurans including Xenopus, we were able to obtain R. temporaria oocytes with a fully developed karyosphere outside the breeding season, namely in December–January. We observed pronounced changes in the chromosomal apparatus of oocytes with a double injection of human chorionic gonadotropin (hCG) at a dose of 500 IU. In this case, chromosomes undergo compaction and aggregation, leading to the formation of a characteristic chromosomal “knot” (karyosphere), the morphological features of which corresponded to those in R. temporaria oocytes at the beginning of the natural breeding season. Based on the proposed protocol for the use of hCG for out-of-season stimulation of oogenesis in R. temporaria, it can be further refined to obtain more stable results and improve the quality of oocytes.

Captive breeding in the endangered alpine tree frog, Litoria verreauxii alpina.

Amphibians are experiencing dramatic worldwide declines and many species are reliant on captive breeding programs to ensure continued survival. However, captive breeding in amphibians is not always successful because many species, especially ones in decline, have particular and specific breeding needs. The endangered alpine tree frog, Litoria verreauxii alpina, has never been bred in captivity before. Due to its dramatic declines across the Australian Alps caused by the global pandemic chytridiomycosis, the species is a potential candidate for captive assurance colonies, which rely on captive breeding. For this study we tested hormone induction using two hormones that have had some success in other amphibian species, to no avail. We then tried outdoor breeding mesocosms during the winter/spring at temperatures similar to their natural breeding season, which was successful. Sixty-five percent of the egg masses laid successfully hatched tadpoles. Females laid more than one clutch over the experiment indicating either a shorter than annual ovulation cycle, or that females are capable of partial ovulation during breeding events. Outdoor breeding mesocosms are a possibility outside the native climate of a species, provided that temperatures overlap with their natural environment. Here, we highlight that troubleshooting is essential before embarking on a captive breeding program of a species that has not been bred before. Hormonal induction of breeding is not always successful; therefore, outdoor mesocosms might be required to achieve healthy tadpoles.

Effects of Dietary Flaxseed Oil and Ascorbic Acid on the Reproductive Performance of South African Indigenous Sheep

The current study aimed to evaluate the effects of the dietary inclusion of flaxseed oil and ascorbic acid on the reproductive performance of South African indigenous sheep (Ovis aries). Twenty-two matured South African indigenous rams (eight BaPedi, nine Zulu, and five Namaqua Afrikaner; age of 6 years and body weight of 64.4 ± 1.6 kg) were randomly assigned into five treatment groups (NC (standard diet), PC (basal diet), FO (5% flaxseed oil), AA (4% ascorbic acid), and FO + AA (5% flaxseed oil + 4% ascorbic acid)). Semen samples were collected during the natural breeding season using an artificial vagina. Semen samples were evaluated for macroscopic (semen volume, pH, and sperm concentration) and microscopic (morphology, malondialdehyde, membrane permeability, and sperm cell motility parameters) characteristics. Eighty-one South African indigenous ewes were synchronized using controlled intravaginal drug devices impregnated with 0.3 g progesterone for 10 days and then injected with 300 IU of Pregnant Mare Serum Gonadotropin. These ewes were then hand-mated. The data were subjected to the General Linear Model (GLM) in Minitub® 2017. A cross-tabulated Chi-Squared (χ2) test was used to track the frequencies of the conception rate. Treatment means were separated using Student’s Least Significant Difference (LSD) and considered significantly different when the p-value was less than 0.05. No considerable breed effect was observed for semen volume, semen pH, sperm concentration, intact membrane, and non-intact membrane. FO + AA led to higher semen volume (1.05 ± 0.06 mL), intact sperm membranes (88.83 ± 1.27%), and low malondialdehyde levels (0.37 ± 0.04 nmol/mL) in comparison to other treatment groups. Total sperm motility was higher in FO + AA (95.81 ± 0.80%) compared to FO, AA, PC, and NC. The conception rate was higher in FO (94%), AA (94%), and FO + AA (100%) when compared to NC (71%) and PC (79%). The testosterone concentration did not differ among PC, FO, AA, and FO + AA. It was concluded that the inclusion of 5% flaxseed oil (FO and FO + AA) improves both the semen quality and the conception rate of South African indigenous sheep.

Incaptive culturing of Goldfish and Koi carp in Punjab, Pakistan

This study observed the captive culture of Carassius auratus and Cyprinus carpio in the Potohar region, Punjab, Pakistan. Captive culturing of both species was conducted up to one-month post hatchlings. The sample size was 180 (90 goldfish and 90 koi carp) and kept in cemented raceways under optimum conditions. The study period was from October 2019 to April 2020. The natural breeding season for Carassius auratus and Cyprinus carpio was from 25th January to 5th February and the end of February to 11th March, respectively. The induced breeding duration for goldfish and koi carp was from 6th to 25th February and for koi carp from 12th to 30th March, respectively. For artificial breeding, the synthetic hormone Ovaprim C was injected intra-peritoneally at 0.5 ml/kg and 0.1 ml/kg body weight of females and males, respectively. Both the species were fed a 30% crude protein diet. Hydrilla plants were used for the adhesion of eggs. The female to male ratio was 2:3. Approximately 4500 to 5000 eggs of goldfish and 17000 to 18000 eggs of koi carp were laid. Fertilization, hatching, and survival rates observed for both were approximately 74%, 57% and 42.5%, respectively.

Variation of heat flux and its effect on the reproduction of four major Chinese carp

Abstract River heat flux (HF) regime has been significantly affected by anthropogenic activities and climate variation, and it is of great significance to deeply explore intrinsic driving mechanisms and ecological effects. This study uses the middle reaches of the Yangtze River as its research area and, by constructing the wavelet model and the IHA-RVA model, quantifies the evolution mechanism and internal law of ‘flow- water temperature (WT) – HF’ over the past four decades and investigates the effects of Three Gorges Dam on the ecological reproduction of ‘four major Chinese carp’. The results show that, (1) flow and WT have three change cycle scales; the overall hydrologic variations of flow and WT were 64% and 62%, respectively, close to high variation. (2) The overall HF shows a decreasing trend from 1983 to 2019, with significant changes in HF in spring and winter regulated by the Three Gorges Reservoir; the basin flow-WT-HF relationships exhibit a hysteretic pattern, with the maximum WT occurring one month after the peak HF and flow. (3) The ‘four major Chinese carp’ natural breeding season is closely related to the time when the WT reaches 18 °C; HF is a vital habitat factor that influences fish spawning and reproduction.

Cryopreservation of small ruminant semen made simple

Successful cryopreservation of small ruminant semen is critical to prosperous artificial insemination programs. Semen collection with the intent to cryopreserve is achieved by either via the use of an artificial vagina or an electroejaculator. Semen collected during the natural breeding season most often has the best prefreezing and postthawing viability. Ejaculates from rams and bucks can be preserved using commercially available OptiXcell II extender and freezing via ‘floatation over liquid nitrogen technique‘ for a short interval.

Comparison of the effect of Gonadotropin-Releasing Hormone and Human Chorionic Go3nadotropin on Reproductive Performance of Romanov Ewes During the Natural Breeding Season.

Low pregnancy rate is an important issue in small ruminants. Superovulation protocols would serve as the possible techniques to increase the pregnancy rate in small ruminants due to the use of synthetic hormones. The present study aimed to assess the effect of gonadotropin-releasing hormone (GnRH) and Human chorionic gonadotropin (hCG) after artificial insemination (AI) on the pregnancy rate of Romanov ewes breed. To this end, estrus was synchronized in 140 ewes using a controlled internal drug-releasing (CIDR) device. Following CIDR removal, all ewes received 400 IU Pregnant Mare's Serum Gonadotropin (PMSG). The ewes were assigned to three groups (control, n=20; experimental groups, each of them was divided into three subgroups, n=20). The first group in each experiment was assumed as control. The second and third groups received three levels of GnRH, (400, 500, and 600 µg, n=20) and hCG (200, 300, and 400 IU) administered by the intramuscular (IM) route. As soon as the signs of estrus were observed, the ewes were isolated from the others and artificially inseminated with fresh sperm using the transcervical method. The results indicated that there were no significant differences between experimental groups in ovulation and pregnancy rates on days 33 and 90 post-AI (P>0.05). The highest twin birth rate (20%) was recorded in the hCG group (300 IU), as compared to GnRH and control groups (P<0.05). The effect of GnRH and hCG on the plasma concentration of progesterone was significant (P<0.01). The groups significantly differed in fecundity rate (Single birth, twin birth, and triple birth) (P<0.05), all of these parameters were significantly higher in the hCG 300 IU group. It can be concluded that the injection of 300 IU of hCG hormone after insemination improves pregnancy rate in ewes.

Effect of advancing the breeding season on reproductive performance of dromedary camels

This study examines the effect of advancing the breeding season on the reproductive performance of dromedary camels under an intensive management system. Using a synchronization protocol, timed natural mating in female camels was carried out either in September (2 months ahead of the natural breeding season, n = 182) or December (peak breeding season, n = 115). The ovarian responses (size of the dominant follicle at the time of mating and ovulation), pregnancy rate, and pregnancy losses were evaluated using ultrasonography. Blood samples were collected after mating to assess progesterone concentrations by RIA. The libido of male camels (n = 13) was evaluated objectively. Results showed that the percentage of female camels with an optimal sized follicle (11–17 mm) for breeding at the time of mating was lower in September compared to December (81.9 vs 91.3%, P = 0.03). The libido of male camels was lower in September than in December (P <0.001). The ovulation rate (86.3 vs 93.9%, P = 0.04), size of the ovulated follicle (12.7 ± 0.1 vs 13.7 ± 0.2 mm, P <0.001), pregnancy rates on Day 14 (47.8 vs 72.2%, P <0.001) and Day 90 (38.5 vs 60.9%, P <0.001) after mating was lower in September compared to December. However, pregnancy loss was not affected between months (15.7 vs 19.5%, P = 0.3). Among pregnant camels, the progesterone concentrations on Days 6, 8, 10, 12 and 14 after mating were lower in September as compared to December (P <0.001). In non-pregnant camels, the progesterone concentrations on Days 6, 8 and 10 after mating were also lower in September as compared to December (P <0.001). In conclusion, advancing the breeding season by two months, significantly affects the reproductive performance of dromedary camels, yet, acceptable pregnancy rates can be achieved.

Pointing Out Some Issues Regarding Reproduction Management in Murciano-Granadina Goats.

Simple SummaryThe hypothesis of this experiment proposes that it could be possible to identify pregnant goats through maximum progesterone milk levels at any time in the pregnancy, and that there is an optimal moment to apply a lactation inhibitor to dry off lactating goats. The maximum progesterone concentration in milk varied depending on the season of the year, and those concentrations were similar for pregnant and non-pregnant goats, but significantly higher in the case of gestating goats with four foetuses, for which it would be possible to distinguish the pregnancy. The milk yield of goats at mating does not affect fertility until a value of at least 3250 mL/day. If using lactation inhibitors, their application up to the 10th week post-mating would be optimal for drying off lactating goats.Two of the most important problems in high-yielding dairy goat farms are early and accurate pregnancy diagnosis and the appropriate dry off of lactating does before the next kidding. The hypothesis posits that it could be possible to identify pregnant does through maximum progesterone milk levels at any time during the pregnancy, and that there is an optimal time to apply a lactation inhibitor to help dry off lactating does. Therefore, 114 Murciano-Granadina breed goats were used, from which 74 goats were inseminated at week 20 of lactation and samples of milk from pregnant and non-pregnant goats were taken at two-week intervals. The average maximum progesterone milk levels were higher outside the natural breeding season (40° latitude) than in the breeding season (11.6 ± 1.13 vs. 8.6 ± 1.02 ng/mL), although the levels from pregnant and non-pregnant goats were similar (10.85 ± 1.3 vs. 9.74 ± 1.6 ng/mL), except in the case of pregnancy with four foetuses (12.5 ± 1.3 ng/mL). Milk yield at mating does not affect fertility until a value of at least 3250 mL/day. Pregnancy started to affect milk yield up to the +7th week and was 59.9% lower in the +10th week after mating, so the use of lactation inhibitors could be more effective from this latter week. In conclusion, the results show that it is not possible to detect gestation in goats reliably through the maximum concentration of progesterone in milk at any time during lactation, except in the case of goats gestating four foetuses, that the milk yield of goats at mating does not affect fertility until a value of at least 3250 mL/day, and that from the 10th week post-mating, the application of lactation inhibitors would be optimal.

Evaluation of seasonal breeding of the domestic canary (Serinus canaria) in an artificial environment

ABSTRACT: The domestic canary (Serinus canaria) has been bred for hundreds of years to improve the quality of its plumage and its song. Reproduction in this species occurs seasonally, stimulated by a gradual increase in day length. Although, the occurrence of seasonal breeding in canaries is well known, whether canary reproduction can be manipulated remains unknown. Our objective was to determine whether the conditions of captivity (photoperiod and temperature) can be adjusted to enable canaries to reproduce outside of their natural breeding season. Thirty days before the natural breeding season, canary pairs were assigned and separated into three different groups: External Control (housed outdoors under ambient conditions), Artificial Control (housed artificially indoors under conditions similar to the external conditions), and Artificial Altered (housed artificially indoors for five months, with the photoperiod gradually manipulated to simulate that of the natural breeding season) groups. The number of clutches laid was greater in the Artificial Control than in External Control; however, more birds hatched in the External Control. In the Artificial Altered group, the beginning of the breeding season was delayed when the same parameters were used. Although, further research is needed, this study presents new data to assist in the development of protocols that entail gradual changes in environmental conditions to try to reduce the impacts of the processes on animal welfare.

141 Diet supplementation alters oocyte lipid content and developmental competence in mares

The equine oocyte is dense in lipids, which may serve as an energy source for oocyte maturation and later embryonic development. However, the association between lipid content and fertility remains to be determined, as does the extent that diet can modify oocyte lipids. We hypothesised that diet supplementation can alter the oocyte lipid profile and subsequent developmental potential. In Study 1, we examined if oocyte triglyceride (TG) relative abundance was affected by dietary supplementation. Mares (16–22 years, n=9) were fed grass/alfalfa hay and supplemented daily with a combination of commercially available feed additives designed to promote equine wellness and fertility [Equine GI™ (147g daily), Potency® (28g daily), Motility Plus® (23g daily), Healthy Weight Oil (60mL daily), Platinum Performance Inc.]. Oocytes were collected from the mares before (PRE) and after ≥8 weeks (POST) of supplementation during the natural breeding season. In Study 2, we compared oocyte developmental potential after injection of sperm into oocytes obtained from mares supplemented for ≥8 weeks with the same additives (ADD, 18–24 yr, n=5) or from a similar group of mares supplemented with a grain control diet (450g of grain mix and 60ml of corn oil daily, GRN, 19–23 years, n=5). Oocytes were collected from dominant follicles (≥35mm) during oestrus and at 20±2 h after induction of follicular maturation. In Study 1, oocytes were denuded of cumulus cells after collection, snap frozen, and assessed for TG composition by nontargeted liquid chromatography-mass spectrometry using an Acquity UPLC system (Waters). In Study 2, recovered oocytes were placed in maturation medium for 22±2h before being injected with sperm from one stallion, and blastocyst formation was assessed in 7 or 8 days. A total of 100 annotated TG species were identified. Normalized peak areas for PRE and POST oocyte TG were compared using two-tailed, paired t-tests. Blastocyst development rates were compared by Fisher’s exact test. Relative abundance of 71 TG species differed (P ≤ 0.05) between PRE and POST; all TG species as well as total relative abundance of TG were higher in oocytes from PRE compared with POST. Blastocyst rates per sperm-injected oocyte were greater (P=0.03) for ADD (40%, 6/15) than for GRN (5%, 1/19). Dietary supplementation of the complex mix of nutrients to middle-aged and older mares resulted in reduced relative abundance of TG in oocytes and improved developmental potential. We determined that oocyte lipid content can be modified through diet. The extent that diet supplementation improved oocyte competence by altering the lipid profile is still to be determined.

Seasonal variations in quantitative and qualitative sperm characteristics in fertile and subfertile stallions.

Horses are seasonal breeders with a natural breeding season beginning in spring and extending through midsummer. In this study, quantitative and qualitative parameters of chilled stallion semen were compared between fertile and subfertile stallions and between the breeding and the non-breeding season. Semen quality parameters compared included ejaculate volume, sperm concentration, total sperm number, sperm morphology, and computer-assisted semen analysis (CASA)-derived sperm movement characteristics obtained from two groups of warmblood stallions (; four fertile stallions and four subfertile stallions), which differ in the seasonal pregnancy rate 80 %–90 % (fertile) vs. 40 %–60 % (subfertile). A total of 64 ejaculates were collected from the stallions (; four in the breeding season and four in the non-breeding season of each stallion). No significant differences in the semen quality parameters between the fertile and the subfertile stallions in the non-breeding season were observed. However, in the breeding season the proportion of morphologically normal sperm, total motility, progressive motility, average path velocity (VAP), and curvilinear velocity (VCL) were significantly higher in the fertile group () when compared with the subfertile group. In addition, a significant seasonal variation in the proportion of morphological normal sperm was found in the fertile group between the breeding and the non-breeding season (). Moreover, significant seasonal variations () in CASA parameters of mean VAP, straight line velocity (VSL), and beat-cross frequency (BCF) were observed in the fertile and the subfertile stallions, which tended to be lower in the non-breeding season. In conclusion, differences between the fertile and the subfertile stallions were observed only in the breeding season, and a few of CASA-derived parameters seemed to be significantly lower during the non-breeding season in both the fertile and the subfertile stallions.

PSXIII-40 Superovulation and oocyte recovery rates in CIDR synchronized goats treated with PGF2α, FSH and GnRH during breeding season

Abstract Embryo transfer is an advanced technology and has great potential in increasing desired livestock without physical boundaries. Multiple embryos can be produced in-vitro or in-vivo using semen from desired males. Early stage embryos (zygotes) are also ideal tools for genome-editing to alter the desired traits. However, the small number of mature oocytes produced each cycle is a major limitation of this technology. Scientists have used drugs to stimulate follicles to increase the number of oocytes/embryos. Extra oocytes/embryos produced can be cryopreserved or shipped to distant locations, to disseminate genetics around the world, at a fraction of cost. Embryos produced from superovulated animals can be transferred in recipient does, to produce desired number of animals. The goal of this study was to estimate the rate of ovulation and oocyte recovery after drug induced superovulation in goats. Eight cycling does were synchronized for estrus, using intravaginal CIDR dispensers, for 11 days during natural breeding season. Each doe was superovulated with a total of 200 mg of pFSH administered intramuscularly in 6 dosages (50 mg, 30 mg, and 20 mg given twice a day at 12 h interval) starting 48 h prior to CIDR removal. Each doe also received 8 mg of PGF2α, 24 h prior to CIDR removal, and 86 µg of GnRH 24 h after CIDR removal. The superovulatory response was evaluated by counting corpus luteum (CL) on each ovary after 48 h of GnRH injection. The ovulated oocytes were recovered surgically from oviducts by flushing with TL Hepes medium. Mean and standard deviation of CL calculated was 10.86±6.34 and 11.67±6.15 CL per doe (n = 8) in left and right ovaries, respectively. Similarly, mean and SD of mature oocytes recovered was 7.67±6.50 and 9.67±5.10 for left and right oviducts, respectively. Average percent recovery of oocytes was 71.23 % / doe.

21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P&amp;lt;0.05. Sperm motility and membrane integrity were significantly higher (P&amp;lt;0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P&amp;lt;0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

Reproductive seasonality in Saanen goats kept under tropical conditions.

This study aimed to verify the reproductive seasonality in Saanen goats from distinct parity orders (nulliparous, primiparous, and pluriparous) throughout an entire year in the Southeast Region of Brazil. In addition, it was also verified the influence of environmental indexes, metabolic profile, body weight, and social interactions on the reproductive seasonality of these animals. Forty Saanen goats kept under intensive system at latitude 22° 52' 30″ s and tropical photoperiod (11-13h of light per day) were used. Every 15days, blood from 24 animals (8 animals per group) was collected for assessment of serum progesterone levels. Monthly, environmental data (rainfall index, maximum and minimum temperature, and time of natural daylight) and blood were collected to evaluate the metabolic profile. Weighting and body condition score measurement were also carried out every 2months. There was no difference in body weight and body condition score in primiparous and pluriparous goats. Nulliparous goats showed natural breeding season in the winter season while the primiparous and pluriparous goats remained cyclical in autumn and winter season. Regardless of the parity order, all goats showed a seasonal anestrus in the spring season. From December, social interactions were able to reestablish, in distinct degrees, the cyclical state in the goats. Likewise, environmental and weight indexes had low and moderate influence, respectively, on the reproductive seasonality in the Saanen goats kept under tropical conditions.

Effect of Melatonin Implants during the Non-Breeding Season on the Onset of Ovarian Activity and the Plasma Prolactin in Dromedary Camel.

To examine a possible control of reproductive seasonality by melatonin, continual-release subcutaneous melatonin implants were inserted 4.5 months before the natural breeding season (October–April) into female camels (Melatonin-treated group). The animals were exposed to an artificial long photoperiod (16L:8D) for 41 days prior to implant placement to facilitate receptivity to the short-day signal that is expected with melatonin implants. The treated and control groups (untreated females) were maintained separately under outdoor natural conditions. Ovarian follicular development was monitored in both groups by transrectal ultrasonography and by plasma estradiol-17β concentrations performed weekly for 8 weeks and then for 14 weeks following implant insertion. Plasma prolactin concentrations were determined at 45 and 15 days before and 0, 14, 28, 56, and 98 days after implant insertion. Plasma melatonin concentration was determined to validate response to the artificial long photoperiod and to verify the pattern of release from the implants. Results showed that the artificial long photoperiod induced a melatonin secretion peak of significantly (P < 0.05) shorter duration (about 2.5 h). Melatonin release from the implants resulted in higher circulating plasma melatonin levels during daytime and nighttime which persisted for more than 12 weeks following implants insertion. Treatment with melatonin implants advanced the onset of follicular growth activity by 3.5 months compared to untreated animals. Plasma estradiol-17β increased gradually from the second week after the beginning of treatment to reach significantly (P < 0.01) higher concentrations (39.2 ± 6.2 to 46.4 ± 4.5 pg/ml) between the third and the fifth week post insertion of melatonin implants. Treatment with melatonin implants also induced a moderate, but significant (P < 0.05) suppressive effect on plasma prolactin concentration on the 28th day. These results demonstrate that photoperiod appears to be involved in dromedary reproductive seasonality. Melatonin implants may be a useful tool to manipulate seasonality and to improve reproductive performance in this species. Administration of subcutaneous melatonin implants during the transition period to the breeding season following an artificial signal of long photoperiod have the potential to advance the breeding season in camels by about 2.5 months.

Time- and dose-related effects of a gonadotropin-releasing hormone agonist and dopamine antagonist on reproduction in the Northern leopard frog (Lithobates pipiens)

Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone release to control ovulation and spermiation in vertebrates. Dopamine (DA) has a clear inhibitory role in the control of reproduction in numerous teleosts, and emerging evidence suggests that similar mechanisms may exist in amphibians. The interactions between GnRH and DA on spawning success and pituitary gene expression in the Northern leopard frog (Lithobates pipiens) were therefore investigated. Frogs were injected during the natural breeding season with a GnRH agonist [GnRH-A; (Des-Gly10, D-Ala6, Pro-NHEt9)-LHRH; 0.1μg/g and 0.4μg/g] alone and in combination with the dopamine receptor D2 antagonist metoclopramide (MET; 5μg/g and 10μg/g). Injected animals were allowed to breed in outdoor mesocosms. Time to amplexus and oviposition were assessed, and egg mass release, incidences of amplexus, egg mass weight, total egg numbers and fertilization rates were measured. To examine gene expression, female pituitaries were sampled at 12, 24 and 36h following injection of GnRH-A (0.4μg/g) alone and in combination with MET (10μg/g). The mRNA levels of the genes lhb, fshb, gpha, drd2 and gnrhr1 were measured using quantitative real-time PCR. Data were analyzed by a two-way ANOVA. Both GnRH-A doses increased amplexus, oviposition and fertilization alone. Co-injection of MET with GnRH-A did not further enhance spawning success. Injection of GnRH-A alone time-dependently increased expression of lhb, fshb, gpha and gnrhr1. The major effect of MET alone was to decrease expression of drd2. Importantly, the stimulatory effects of GnRH-A on lhb, gpha and gnrhr1 were potentiated by the co-injection of MET at 36h. At this time, expression of fshb was increased only in animals injected with both GnRH-A and MET. Spawning success was primarily driven by the actions of GnRH-A. The hypothesized inhibitory action of DA was supported by pituitary gene expression analysis. The results from this study provide a fundamental framework for future time- and dose–response investigations to improve current spawning methods in amphibians.

Synchronization estrus in sheep Merinos of Palas in natural breeding season, using Chrono-Gest of type sponges (FGA)

Synchronization estrus in sheep Merinos of Palas in natural breeding season, using Chrono-Gest of type sponges (FGA)