Enhancement of the solubility and oral bioavailability of altrenogest through complexation with hydroxypropyl-ß-cyclodextrin.

Altrenogest (ALT), a synthetic progestogen, serves a critical role in estrus synchronization among animals like gilts and mares. However, its practical application in animal husbandry is hampered due to its poor solubility and limited oral bioavailability. To address this challenge, a solvent evaporation method was employed to create an inclusion complex of ALT with hydroxypropyl-ß-cyclodextrin (ALT/HP-ß-CD). The formation of this inclusion complex was confirmed by scanning electron microscopy, power X-ray diffraction, differential scanning calorimetry, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and docking calculations. In addition, we further conducted pharmacokinetic investigation involving gilts, comparing ALT/HP-ß-CD inclusion complex to an ALT oral solution. The physicochemical characterization results unveiled a transformation of ALT's crystal morphology into an amorphous state, with ALT effectively entering the cavity of HP-ß-CD. Compared with ALT, the solubility of ALT/HP-ß-CD inclusion complex increased by 1026.51-fold, and its dissolution rate demonstrated significant improvement. Pharmacokinetic assessments further revealed that the oral bioavailability of ALT/HP-ß-CD inclusion complex surpassed that of the ALT oral solution, with a relative bioavailability of 114.08 %. In conclusion, complexation with HP-ß-CD represents a highly effective approach to improve both the solubility and oral bioavailability of ALT.

Effect of a combination of altrenogest and double PGF2α administrations on farrowing variation, piglet performance and colostrum IgG.

The variation of gestation length in sows leads to difficulties performing farrowing supervision. The present study was performed to investigate whether oral administration of altrenogest until 112 days of gestation and double administration of PGF2α at 113 days of gestation can synchronise the onset of parturition in sows and minimise deleterious effects on the incidence of stillbirths and colostrum quality. Additionally, the effects of synchronised farrowing on colostrum yield and piglet birth weight, colostrum intake and survival rate of piglets until seven days of postnatal life were also investigated. In total, 193 Landrace x Yorkshire crossbred sows were randomly allocated according to parity number into two groups, i.e. control (n = 95) and treatment (n = 98). The control sows were allowed to farrow naturally. The treatment sows were orally administered 20 mg per day of altrenogest for four days from 109 to 112 days of gestation and were administered PGF2α twice on day 113 of gestation. Individual body weight at birth and 24 h after birth of piglets in all litters were determined in both control (n = 1609) and treatment (n = 1707) groups. Colostrum consumption of all piglets, colostrum yield, colostrum IgG and serum progesterone of sows were determined. On average, the total number of piglets born per litter were 17.0 ± 3.1. The proportion of sows farrowed before 114 days of gestation was higher in the control than the treatment group (8.4% and 2.0%, respectively, P = 0.05) and 92.8% of sows in the treatment group farrowed on day 114 of gestation. The percentage of stillborn piglets per litter did not differ significantly between control and treatment groups (4.5% and 4.6%, respectively). Colostrum yield of sows did not differ between control and treatment groups (5.52 ± 0.13 and 5.28 ± 0.12 kg, respectively, P = 0.174). However, colostrum intake of piglets was lower in the treatment than the control group (354.7 ± 6.6 and 381.2 ± 7.0 g, respectively, P = 0.012). Colostrum IgG was higher in the control than the treatment group (41.2 ± 1.1 and 37.3 mg per ml, P = 0.013). In conclusion, altrenogest treatment from 109 to 112 days and double administrations of PGF2α on day 113 of gestation can control gestation length in sows. No deleterious effects of this protocol on the incidence of stillbirths and sow colostrum yield were detected. However, piglet colostrum intake and colostrum IgG were compromised. Thus, care of newborn piglets in the treatment group should be considered.

Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period.

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and is causal to seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. Postpubertal gilts (n = 42) were artificially inseminated during behavioral estrus (n = 28) or were kept cyclic (n = 14), and randomly assigned to thermal neutral (TN; 21 ± 1 °C) or diurnal HS (35 ± 1 °C for 12 h/31.6 ± 1 °C for 12 h) conditions from day 3 to 12 postestrus (dpe). Seven of the inseminated gilts from each thermal treatment group received ALT (15 mg/d) during this period. Using quantitative PCR, transcript abundance of HSP family A (Hsp70) member 1A (HSPA1A, P = 0.001) and member 6 (HSPA6, P < 0.001), and HSP family B (small) member 8 (HSB8, P = 0.001) were increased while HSP family D (Hsp60) member 1 (HSPD1, P = 0.01) was decreased in the endometrium of pregnant gilts compared to the cyclic gilts. Protein abundance of HSPA1A decreased (P = 0.03) in pregnant gilt endometrium due to HS, while HSP family B (small) member 1 (HSPB1) increased (P = 0.01) due to HS. Oral ALT supplementation during HS reduced the transcript abundance of HSP90α family class B member 1 (HSP90AB1, P = 0.04); but HS increased HSP90AB1 (P = 0.001), HSPA1A (P = 0.02), and HSPA6 (P = 0.04) transcript abundance irrespective of ALT. ALT supplementation decreased HSP90α family class A member 1 (HSP90AA1, P = 0.001) protein abundance, irrespective of thermal environment, whereas ALT only decreased HSPA6 (P = 0.02) protein abundance in TN gilts. These results indicate a notable shift of HSP in the porcine endometrium during the peri-implantation period in response to pregnancy status and heat stress.

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and causes seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. We evaluated the abundance of HSP90, HSP70, HSP60 and HSPB in the porcine endometrium during the peri-implantation period. We demonstrate how a physiological event such as pregnancy and an environmental stressor such as HS, individually and in combination, alter the endometrial abundance of these HSP. Moreover, supplementation of pregnant gilts subjected to HS with ALT also altered the abundance of these HSP in the porcine endometrium.

Altrenogest treatment reduces the stress response of three-year-old warmblood mares during their initial equestrian training.

Horse mares are frequently treated with the progestin altrenogest with the aim to suppress estrous behavior and its negative impact on equestrian performance. Progestogens, however, also have sedative effects in males, and females of different species. The aim of our study was therefore to investigate altrenogest-induced changes in the stress response of female horses during initial equestrian training. Three-yr-old Warmblood mares were randomly assigned to treatment with altrenogest (ALT; 0.044 mg/kg once daily; n = 6) or sunflower oil (CON; n = 5) for 12 wk during training. At predefined steps of the training program (free movement, lunging without and with side reins, lunging with saddle, mounting of a rider, free riding, riding by an unfamiliar rider) salivary cortisol concentration, and heart rate were determined from 60 min before to 120 min after training. The same procedures were performed during repeated gynecologic examinations and 2 novel object tests. Bodyweight and body condition scores (BCS) were assessed at 4-wk intervals. During all training units, salivary cortisol concentration and heart rate increased (P < 0.001), but the increase was smaller in group ALT mares (time x treatment P < 0.001). Gynecologic examinations and novel object tests induced a much smaller increase in cortisol and heart rate (P < 0.001) than equestrian training with no difference between groups ALT and CON. Initially, bodyweight, and BCS decreased during training. The subsequent increase was larger in group ALT vs CON (time x treatment P < 0.05). In conclusion, altrenogest reduced the stress response of 3-yr-old mares to equestrian training. The use of altrenogest during equestrian competitions should therefore be reconsidered.

Control of parturition in hyperprolific sows by using altrenogest and double administrations of PGF2α.

The aim of the present study was to develop a protocol to reduce the variation in gestation length and synchronise the onset of parturition in sows by using altrenogest in combination with double administrations of prostaglandin F2alpha (PGF2α). In total, 188 Landrace x Yorkshire crossbred sows with parity numbers 3.1 ± 1.6 were included in the experiment. The sows were classified into two groups: CONTROL (n = 94) and TREATMENT (n = 94). CONTROL sows were allowed to farrow naturally, and TREATMENT sows were orally administered 20 mg/day of altrenogest starting when they entered the farrowing house (107.0 ± 2.0 days) until 113 (TREAT-113, n = 18), 114 (TREAT-114, n = 29) and 115 (TREAT-115, n = 47) days of gestation. The altrenogest-treated sows were administered PGF2α twice 6 h apart at 24 h after the withdrawal of altrenogest. The litters were randomly selected (25 and 26 litters from CONTROL and TREATMENT groups, respectively) to determine individual body weight at birth and at 24 h after birth. Gestation length of sows that farrowed naturally averaged 115.1 days (range 111-118), whereas gestation length of altrenogest-treated sows averaged 115.1-116.3 days (range 114-118). The colostrum yield of sows averaged 4.25 ± 1.19 kg and was not affected by the treatment (P > 0.05). Colostrum IgG in the CONTROL group was higher than in the TREAT-114 and TREAT-115 groups (P < 0.05) but did not differ significantly compared to the TREAT-113 group. The proportion of sows that farrowed during working hours (0700-1700 h) in the TREAT-113 group (72.3%) tended to be higher than in the CONTROL (46.4%, P = 0.053). The interval from the last altrenogest treatment until farrowing in the TREAT-113 group was longer than in the TREAT-114 and TREAT-115 groups (62.8, 40.7 and 34.6 h, respectively, P < 0.05). Similarly, the intervals from the first PGF2α administration to the onset of parturition in the TREAT-113 group (38.8 ± 3.8 h) was longer than TREAT-114 (21.9 ± 3.5 h, P = 0.002) and TREAT-115 (25.5 ± 3.7 h, P = 0.016) groups. However, the incidence of stillbirths in the TREAT-113, TREAT-114 and TREAT-115 groups was higher than in the CONTROL (16.4, 17.2, 11.8 and 5.8%, respectively, P < 0.05). In conclusion, altrenogest supplementation in combination with double administrations of PGF2α can reduce the variation in gestation length and synchronise the onset of parturition in sows. However, its side effects on the incidence of stillbirths should be considered.

Effects of increased ambient temperature and supplemental altrenogest before pregnancy establishment in gilts.

Heat stress (HS) mitigation strategies are critically needed to combat the substantial economic effects on animal agriculture. The manifestations of seasonal infertility include delayed puberty onset, reduced conception rates, decreased litter size, and increased wean to estrus interval. To assess the effects of HS during early gestation and evaluate the benefit of supplemental altrenogest (ALT) as a mitigation strategy, 30 crossbred postpubertal gilts (157 ±â€…11 kg body weight) were subjected to estrous synchronization via 14 d oral administration of ALT. Artificial insemination during estrus was performed, and gilts were then placed into one of four treatment groups: HS (35 ±â€…1 °C for 12 h/31.60 ±â€…1 °C for 12 h) with (HSALT, n = 7) or without (HSCON, n = 7) 15 mg/d ALT supplementation or thermal neutral (TN; 20 ±â€…1 °C) conditions with (TNALT, n = 8) or without (TNCON, n = 8) 15 mg/d ALT supplementation until 12 d post-estrus (dpe). Administrating ALT occurred at 0600 hours from 3 to 12 dpe, and rectal temperatures (TR) and respiration rates (RR) were recorded. Blood was collected via jugular venipuncture on 0, 4, 8, and 12 dpe. Gilts were euthanized humanely at 12 dpe followed by the collection of ovarian tissue, and uterine flushing for conceptus collection. In HS compared with TN gilts, RR and TR were increased (P < 0.01) but unaffected by ALT supplementation. Feed intake was reduced (P < 0.01) by HS but unaltered by the ALT treatment. Corpora lutea (CL) weight was reduced (P < 0.01) in HSCON gilts when compared with TNCON and HSALT gilts despite progesterone concentrations in serum and luteal tissue not being affected by treatment (P ≥ 0.10). CL diameter was reduced (P ≤ 0.05) in HSALT gilts compared with other treatments. Interleukin-1ß (IL1B) uterine flush concentration was not affected (P > 0.20) by environment or ALT supplementation, although moderate (P = 0.06) interaction between environment and ALT existed, as IL1B concentration in TNALT was increased (P = 0.03) compared with TNCON gilts. While environment did not affect conceptus development (P = 0.90), ALT supplementation advanced conceptus elongation (P < 0.01). Collectively, these data demonstrate that HS may affect luteal development before pregnancy establishment, and ALT increases conceptus elongation by 12 dpe.

Comprehensive LC-HRMS metabolomics analyses for the estimation of environmental inputs of altrenogest in pig breeding.

Altrenogest (ALT), a synthetic progestogen, is used in pig farming for estrus synchronization in gilts. Residues of ALT and its metabolites may reach the aquatic environment via the spread of liquid manure and may present a risk for fish and other higher aquatic organisms due to its endocrine disrupting potential. A pilot study was conducted in which spot urine samples from ALT-treated and non-medicated gilts were collected. We applied LC-HRMS analysis to perform targeted analysis of ALT and known metabolites as well as non-targeted metabolomics analyses to find previously unknown metabolites. The targeted investigation showed that glucuronide conjugates of ALT and its photo-isomerization product are main urinary metabolites of ALT in gilts. Furthermore, an unknown isomerization product of ALT was observed at trace level, whereas ALT and ALT sulfate were not found. The chemometric analysis of non-targeted data revealed a clear difference between ALT-treated gilts and control animals. Furthermore, a hydroxylated ALT glucuronide was identified as highly significant in the ALT-treated group. Additional biomarker annotation and pathway mapping revealed changes in the metabolism of ALT-treated animals which can be explained by ALT's hormonal action. This study illustrates the exceptional potential of LC-HRMS and metabolomics for the detection of potentially new environmental contaminants with high biological activity. Further advantages of the method described are the sampling during routine breeding conditions, a relatively small number of animals required and no particular stress for the animals.

Altrenogest treatment during the last week of lactation on ovarian traits and subsequent reproductive performance of primiparous and multiparous sows.

High-quality follicles result in larger corpora lutea (CL), producing more progesterone, and having a fundamental role in pregnancy maintenance. For some sows, follicular growth takes place during lactation, and follicle selection occurs under a catabolic environment. As altrenogest inhibits follicular development, this study aimed to evaluate follicular growth, CL size, estrus expression, and subsequent reproductive performance of sows treated with altrenogest during the last seven days of a three-week lactation. A total of 81 primiparous and 319 multiparous sows were allocated to two treatments: CONT (control group) and ALT (20 mg of altrenogest/day during the last seven days of lactation). Subsamples of 20 primiparous sows and 97 multiparous were randomly selected to evaluate follicular growth and 26 multiparous sows for serum progesterone analysis at day 21 of gestation. On day 21 of pregnancy, CL measurement was performed by ultrasound. Once in estrus, sows were post-cervically inseminated with pooled semen doses with 1.5 × 109 sperm cells at estrus onset and every 24 h during the standing reflex period. Sows not showing estrus until 10 days after weaning were considered in anestrus. The variables weaning-to-estrus interval, CL size, litter size in the subsequent cycle, and piglet birth weight were evaluated using the GLIMMIX procedure and compared using the Tukey-Kramer test. Anestrus, pregnancy, farrowing, and adjusted farrowing rate were evaluated as binary responses using logistic regression. Follicular size was analyzed as a repeated measure during treatment and after weaning. Treatment was considered as a fixed effect. During the treatment period, follicular size was smaller in ALT sows than CONT sows (3.29 vs. 3.52 mm; P < 0.001). However, after treatment, ALT sows showed a larger follicular size than CONT sows (5.30 vs. 5.03 mm; P ≤ 0.01). There were less ALT sows showing estrus than CONT sows on days three (1.03 vs. 4.57%) and four (55.38 vs. 68.02%) after weaning (P ≤ 0.05), respectively. At 21 days after insemination, ALT sows showed larger CL size and lower CL size variation (P < 0.01) than CONT sows. Anestrus rate, pregnancy rate, farrowing rate, adjusted farrowing rate, litter size in the subsequent cycle, piglet birth weight, litter birth weight, and birth weight variation did not differ between treatments (P ≥ 0.14). In conclusion, altrenogest treatment during the last week of lactation concentrated estrus expression on day five after weaning, larger follicle and CL sizes; however, with no improvement in reproductive performance.

Downregulation of testicular function in the goat by altrenogest.

BACKGROUND: The present study investigated whether the administration of the progestin altrenogest provides noninvasive, temporary, and reversible suppression of gonadal function in the goat as a potential alternative to chirurgical castration, which is related with irreversibility, risks of complications till death of the animal and welfare issues. Eight sexually mature Peacock goats were randomly divided into two groups. The experimental group was administered altrenogest (0.088 mg/kg) orally once daily for 7 weeks. The remaining four goats received an oral glucose solution and served as the control group. After completing the administration period, the reversibility of the medication was evaluated for another 7 weeks (observation phase). The treatment effects were assessed by clinical examination; ultrasound examination of the testes, including one-dimensional grayscale analysis, blood testosterone levels, analysis of semen parameters and libido. At the end of the observation period, the animals were castrated and the testicles were examined histologically. RESULTS: Altrenogest treatment had no significant effect on the physical development of the goats, the sonographic appearance of the testes, the gray values measured in the ultrasound images, or the blood testosterone levels. The effects of treatment on the testicular and semen parameters varied widely in the experimental animals; the testicle volume was significantly lower and the number of pathologically altered sperm in the ejaculate was significantly higher in treated animals. CONCLUSION: These findings indicate that daily altrenogest administration at a dose of 0.088 mg/kg does not reliably suppress gonadal function in the goat.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Outcome of bilateral equid laparoscopic ovariectomies.

OBJECTIVE: To assess outcomes and behavior changes associated with bilateral laparoscopic ovariectomies. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Fifty-one equids. METHODS: Medical records were evaluated from equid bilateral laparoscopic ovariectomies from January 2012 to October 2018 with a potential of 6 months follow-up. Follow-up information obtained by telephone interviews included behavior before and after surgery. Likelihood ratio chi-square tests and odds ratios (OR) with 95% CI were calculated where applicable, with statistical significance at p < .05. RESULTS: Bilateral ovariectomy was performed in 51 cases, with elective (no pathologic ovaries) ovariectomies performed in 41/51 cases. Occasional estrus-like behavior was observed postoperatively in 14/51 (27%) mares, but the behavior was mild and manageable in all cases. There was no age effect on outcome in all bilateral (p = .56) or elective only (p = .36) cases. In 37/41 (90%) elective cases, improvement was observed in the reason for presentation. Some response to altrenogest administration for behavior modification was observed preoperatively in 12/18 (67%) elective cases. Response to altrenogest was not associated with (p = .31) or able to predict a beneficial response to surgery (OR = 5.5; 95% CI = 0.38-78.57; p = .21). CONCLUSION: Response to altrenogest in elective cases may not predict behavioral outcome with ovariectomy. Occasional estrus-like behavior in mares postoperatively was not problematic for any owners. Bilateral ovariectomy is a viable treatment option for owners seeking to alleviate undesirable behavior in mares. CLINICAL SIGNIFICANCE: This study should aid veterinarians and horse owners in case selection for bilateral ovariectomy.

Unravelling androgens in sport: Altrenogest shows strong activation of the androgen receptor in a mammalian cell bioassay.

Altrenogest is a commonly used progestogen for the suppression of oestrus and associated distracting behaviours that interfere with training and performance of female racehorses. The steroid is derived from 19-nor testosterone and is structurally similar to the anabolic androgenic steroid, trenbolone. In this study, the relative androgen potency of altrenogest was determined by a kidney (HEK293) cell androgen bioassay. The HEK293 bioassay shows that in its pure form, altrenogest has a high relative potency compared with testosterone but is not as strong as ß-trenbolone. Our results also show that altrenogest is able to activate the androgen receptor at the concentrations relevant to the administration regime of racehorses and retains its activity ex vivo. Thus, we show unequivocally that altrenogest, a progestogen used widely in female racehorses, acts as a strong androgen in a mammalian cell bioassay.

Comparative pharmacokinetic study of two kinds of altrenogest oral solutions for sows.

This study was to compare the pharmacokinetic characteristics of domestic altrenogest oral solution (DAOS) or imported altrenogest oral solution (IAOS) in healthy sows. A single administration (1 mg/kg body weight) of DAOS or IAOS was performed in sixteen healthy sows according to a two-period crossover design. Plasma concentrations of altrenogest (AT) were measured by high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS) and the concentration-time data of AT was analyzed by WINNONLIN 5.2. It was suggested that the main pharmacokinetic parameters of DAOS and IAOS were as follows: Cmax was 227.59 ± 83.35 ng/mL and 152.83 ± 80.34 ng/mL, Tmax was 1.16 ± 0.52 h and 1.58 ± 0.85 h, t1/2 was 3.63 ± 0.72 h and 3.45 ± 0.63 h, MRT was 5.02 ± 0.79 h and 5.21 ± 0.87 h, AUC0-t was 1050.23 ± 409.80 h·ng/mL and 778.22 ± 397.84 h·ng/mL, and AUC0-∞ was 1060 h·ng/mL and 786 h·ng/mL, respectively. The relative bioavailability of DAOS was 134.9%. Above results indicated that oral DAOS was better absorbed than IAOS, Cmax of DAOS was higher than that of IAOS (p < 0.05). However, there was no significant difference in the main pharmacokinetic parameters between oral DAOS and IAOS (p > 0.05). Our data confirmed that the absorption, fast elimination and bioavailability of DAOS in sows were better than those of IAOS.

Investigations on residual elimination of altrenogest oral solution in pigs and the withdrawal time.

The residual elimination of altrenogest in swine was investigated, preparing for the determination of withdrawal time. The residues of altrenogest in sebum, muscle, liver and kidney were extracted by optimized methods and further analyzed by UPLC-MS/MS. Under experimental conditions, the LOD and LOQ of altrenogest in sebum, muscle, liver and kidney were 0.5 and 1.0 µg/kg, respectively. The recoveries were in the range of 65 and 95% and the inter- and intra-RSD were less than 15%. The established method for the extraction, purification and detection of altrenogest is suitable for the determination of the residue of altrenogest in edible tissues of pigs. It was showed that altrenogest had the highest residual concentration level in liver, followed by kidney, sebum and muscle. Then withdrawal time was set at 9 days. The study provides an effective basis for elimination of altrenogest in swine.

Altrenogest affects expression of galectin-3 and fibroblast growth factor 9 in the reproductive tract of sows.

A synthetic progestin altrenogest (ALT) is used to synchronize the estrus cycle of swine for fixed-time artificial insemination (AI) and has been shown to improve follicular development and reproductive performances in post-weaning sows. However, the effects of ALT treatment on reproductive tracts, including the ovaries, oviducts and uterus have not been yet clarified. In this study, we examined the expression of genes involved in endometrial responses in ALT-treated sows. ALT did not significantly alter luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol profiles in blood compared to untreated control. Quantitative RT-polymerase chain reaction (qRT-PCR) analysis showed that the expression of genes encoding galectin-3 (LGALS3) and fibroblast growth factor 9 (FGF9) was upregulated in the reproductive tracts of ALT-treated sows, including the ovaries, oviducts and uteri. Moreover, ALT treatment induced the expression of FGF9 and galectin-3 proteins, and promoted their localization to the luminal epithelium of the oviducts and uterus. Our findings suggest that the enhancement of reproductive performance shown by ALT-treated sows is associated with the upregulation of galectin-3 and FGF9, which are essential for endometrial receptivity, successful implantation, and pregnancy.

Improving ovulation in gilts using anti-inhibin serum treatment combined with fixed-time artificial insemination.

For successful batch farrowing, porcine oestrus and ovulation must be synchronized using fixed-time artificial insemination (FTAI). However, exogenous gonadotropins, which are currently used in FTAI, negatively affect gilt ovulation. Here, we aimed to improve sexually mature gilt superovulation efficiency using passive immunization against inhibin during FTAI. Altrenogest-treated gilts were challenged with 10 ml anti-inhibin serum (AIS group, n = 6), 1,000 IU pregnant mare serum gonadotropin (PMSG group, n = 6), or 10 ml goat serum (control group, n = 6). Gilts in the AIS and PMSG groups were inseminated according to the FTAI protocol, and gilts in the control group were inseminated during natural oestrus. When PMSG was replaced by AIS during FTAI of gilts, ovulation rate and embryos recovered were significantly greater in the AIS group as compared to the other two groups (p < .05). Especially the average number of 6-8-cell embryos in the AIS group was significantly higher than that in the PMSG group (p < .01). Moreover, the blastocyst number in the AIS group was significantly higher than that in the PMSG group and the control group (p < .05). But there was no significant difference in the blastocyst number between the PMSG group and the control group (p > .05). Besides, plasma levels of estradiol-ß (E2) and progesterone (P4) were significantly greater in the AIS group as compared to the other two groups on Day 23 and D 27, respectively (p < .01). In summary, we devised an improved high-yield FTAI protocol for sexually mature gilts using AIS; this protocol had a greater superovulation efficiency than the FTAI using PMSG.

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Effects of oestrous synchronization with altrenogest in gilts on endometrial and embryonic characteristics.

The use of altrenogest (ALT) supplementation for oestrous synchronization improves subsequent reproductive performance of gilts and sows. However, the causes of this improvement in reproductive performance after ALT treatment are not fully/clearly understood. The objective of this study was to evaluate the effects of ALT supplementation for oestrous synchronization in gilts on the endometrial glands and embryonic development characteristics at 28 days of pregnancy. Pregnant gilts were divided into two experimental treatments: Control (did not receive ALT; n = 9 gilts) and ALT (ALT feeding at 20 mg/day for 18 days; n = 9 gilts). At 28 days of pregnancy, six gilts from each treatment were slaughtered, and reproductive tracts were immediately evaluated. There was no statistical difference (P > 0.05) between treatments regarding ovulation rate, number of embryos, number of vital embryos and number of non-vital embryos. Embryo weight, length and embryonic vesicle weight were lower in ALT treatment compared with Control (P < 0.01), and it was lower in the cervical uterine region compared with apex uterine region, respectively (P < 0.05). Higher values of gland duct area, gland duct perimeter, percentage of the glandular area and total endometrial area were observed in ALT treatment compared with Control (P < 0.05). The use of ALT during 18 days for oestrous synchronization in gilts increased the gland duct area, perimeter and total endometrial area but did not increase the embryo number and embryo size at day 28 of pregnancy.

Supplemental progesterone during early pregnancy exerts divergent responses on embryonic characteristics in sows and gilts.

Progesterone (P4) plays a key role in pregnancy establishment and maintenance; during early pregnancy, P4 stimulates the production and release of uterine secretions necessary for conceptus growth prior to implantation; therefore, exogenous P4 supplementation may improve embryo development. This study evaluated the effects of supplementation during early pregnancy with long-acting injectable progesterone or altrenogest on embryonic characteristics of sows and gilts. Thus, a total of 32 sows and 16 gilts were used. On day 6 of pregnancy sows and gilts were allocated to one of the following groups: non-supplemented; supplemented with 20 mg of altrenogest, orally, from days 6 to 12 of pregnancy; supplemented with 2.15 mg/kg of long-acting injectable progesterone on day 6 of pregnancy. Animals were killed on day 28 of pregnancy, and ovulation rate, embryo survival, embryo weight, crown-to-rump length, uterine glandular epithelium and endometrial vascularization were assessed. Treatments had no effect on pregnancy rate, embryo survival or endometrial vascular density (P > 0.05). Non-supplemented gilts presented larger and heavier embryos compared to gilts from supplemented groups (P < 0.05). Sows in the altrenogest group presented larger and heavier embryos compared to non-supplemented sows and sows supplemented with long-acting injectable progesterone. In conclusion, supplementation of sows and gilts with progestagen from day 6 of pregnancy can be used as a means to improve embryo survival without deleterious effects.

Differentiating between the effects of heat stress and lipopolysaccharide on the porcine ovarian heat shock protein response1.

Heat stress (HS) negatively affects both human and farm-animal health and undermines efficiency in a variety of economically important agricultural variables, including reproduction. HS impairs the intestinal barrier, allowing for translocation of the resident microflora and endotoxins, such as lipopolysaccharide (LPS), from the gastrointestinal lumen into systemic circulation. While much is known about the cellular function of heat shock proteins (HSPs) in most tissues, the in vivo ovarian HSP response to stressful stimuli remains ill-defined. The purpose of this study was to compare the effects of HS or LPS on ovarian HSP expression in pigs. We hypothesized that ovarian HSPs are responsive to both HS and LPS. Altrenogest (15 mg/d) was administered per os for estrus synchronization (14 d) prior to treatment and three animal paradigms were used: (i) gilts were exposed to cyclical HS (31 ± 1.4 °C) or thermoneutral (TN; 20 ± 0.5 °C) conditions immediately following altrenogest withdrawal for 5 d during follicular development; (ii) gilts were subjected to repeated (4×/d) saline (CON) or LPS (0.1 µg/kg BW) i.v. infusion immediately following altrenogest withdrawal for 5 d; and (iii) gilts were subjected to TN (20 ± 1 °C) or cyclical HS (31 to 35 °C) conditions 2 d post estrus (dpe) until 12 dpe during the luteal phase. While no differences were detected for transcript abundances of the assessed ovarian HSP, the protein abundance of specific HSP was influenced by stressors during the follicular and luteal phases. HS during the follicular phase tended (P < 0.1) to increase ovarian protein abundance of HSP90AA1 and HSPA1A, and increased (P ≤ 0.05) HSF1, HSPD1, and HSPB1 compared with TN controls, while HS decreased HSP90AB1 (P = 0.01). Exposure to LPS increased (P < 0.05) HSP90AA1 and HSPA1A and tended (P < 0.1) to increase HSF1 and HSPB1 compared with CON gilts, while HSP90AB1 and HSPD1 were not affected by LPS. HS during the luteal phase increased (P < 0.05) abundance of HSPB1 in corpora lutea (CL), decreased (P < 0.05) CL HSP90AB1, but did not impact HSF1, HSPD1, HSP90AA1, or HSPA1A abundance. Thus, these data support that HS and LPS similarly regulate expression of specific ovarian HSP, which suggest that HS effects on the ovary are in part mediated by LPS.