Corrigendum to "Which factor is more important for the welfare of broiler chickens: Intensity or duration of episodic heat stress?" [J. Therm. Biol. 99 (2021) 102981].

Due to the effects of global warming, there is a predicted increase in the frequency, intensity and duration of heat waves in the future. Little is known of how this could affect the welfare of broiler chickens. Sixty-four broiler chickens were subjected to either high heat stress (HHS; 32oC, 70% RH for 3 h), moderate heat stress (MHS; 30oC, 70% RH for 6 h), or normal conditions (NC: 20oC, 50% RH for 6 h) for two consecutive days. Half the birds had been subjected to anaesthesia and fitted with a body temperature-ID chip placed in the breast muscle. Core body temperature (CBT) was taken during pre-heat stress (PrHS), at the end of 3 h (3HS) and 6 h (6HS) of heat stress using a pocket reader and used to estimate change in CBT (ΔCBT). Surface body temperatures (SBTs) from under the wing (WT), feet (FT), cloaca (CLT) and comb (CT) were also measured, along with blood parameters, feed intake, daily weight gain and mortality. Data were analysed using General Linear Model and simple linear regression. At 3HS, CBT/ΔCBT and all SBTs showed this trend: HHS > MHS > NC (P<0.001). Blood pH, pCO2, iCa, HCO3- and TCO2 showed the same trend: HHS, MHS > NC (P<0.05). Comparing HHS for 3 h with MHS and NC for 6 h showed that CBT/ΔCBT, WT and CLT in HHS, MHS > control (P<0.001) while FT and CT showed a different trend (HHS > MHS > NC, P<0.001). Exposure of broiler chickens to 3 hours of HHS had dramatic effects on core and surface body temperatures. The effects of MHS were initially more modest yet, after a further 3 hours exposure, resulted in an increase in CBT which was close to that which HHS birds experienced after just 3 hours. This illustrates that duration of exposure to heat stress can have a critical effect, achieving similar life-threatening changes in body temperature that were observed under higher levels of heat stress but for half the time.

Which factor is more important: Intensity or duration of episodic heat stress on broiler chickens?

With the current global warming, there is a predicted increase in frequency, intensity and duration of heat waves in future. Little is known of how this could affect the welfare of broiler chickens. Sixty-four broiler chickens were subjected to either high heat stress (HHS; 32 °C, 70% RH for 3 h), moderate heat stress (MHS; 30 °C, 70% RH for 6 h), or normal conditions (NC: 20 °C, 50% RH for 6 h) for two consecutive days. On both days, the temperature-ID chips on all chipped birds were scanned during pre-heat stress (PrHS), end of 3 h (3HS) and 6 h (6HS) of heat stress using a pocket reader. Half of the chip birds' CBT was measured at the end of each hour of heat stress (HS: 1st -3rd hour). Surface body temperatures (SBTs) from under the wing (WT), feet (FT), cloaca (CLT) and comb (CT) were measured. Blood samples, feed intake, daily weight gain and mortality was also monitored. Data was analysed using General Linear Model and simple linear regression. At 3HS, CBT/ΔCBT and all SBTs showed this trend: HHS>MHS>NC (P<0.001). The regression equations to predict ΔCBT in HHS and MHS are ΔCBT = 0.917 + 0.663 h, P<0.05 and ΔCBT = 0.371 + 0.338 h, P<0.05 respectively. Blood pH, pCO2, iCa, HCO3- and TCO2 showed same trend: HHS, MHS > NC (P<0.05). Comparing HHS for 3 h with MHS and NC for 6 h shows that CBT/ΔCBT, WT and CLT in HHS, MHS>control (P<0.001) while FT and CT showed a different trend (HHS > MHS > NC, P<0.001). pCO2, feed intake and daily weight gain showed same trend (HHS, MHS > control). Temperature-ID chip (a less invasive technique) gave CBT/ΔCBT values that corresponded with the degree of heat stress experienced by the birds. Broilers were more tolerant to MHS than HHS after 3 h but MHS for 6 h and HHS for 3 h had similar impact.

Identification of stable genes in the corpus luteum of lactating Holstein cows in pregnancy and luteolysis: Implications for selection of reverse-transcription quantitative PCR reference genes.

In lactating dairy cattle, the corpus luteum (CL) is a dynamic endocrine tissue vital for pregnancy maintenance, fertility, and cyclicity. Understanding processes underlying luteal physiology is therefore necessary to increase reproductive efficiency in cattle. A common technique for investigating luteal physiology is reverse-transcription quantitative PCR (RT-qPCR), a valuable tool for quantifying gene expression. However, reference-gene-based RT-qPCR quantification methods require utilization of stably expressed genes to accurately assess mRNA expression. Historically, selection of reference genes in cattle has relied on subjective selection of a small pool of reference genes, many of which may have significant expression variation among different tissues or physiologic states. This is particularly concerning in dynamic tissues such as the CL, with its capacity for rapid physiologic changes during luteolysis, and likely in the less characterized period of CL maintenance during pregnancy. Thus, there is a clear need to identify reference genes well suited for the bovine CL over a wide range of physiological states. Whole-transcriptome RNA sequencing stands as an effective method to identify new reference genes by enabling the assessment of the expression profile of the entire pool of mRNA transcripts. We report the identification of 13 novel putative reference genes using RNA sequencing in the bovine CL throughout early pregnancy and luteolysis: RPL4, UQCRFS1, COX4I1, RPS4X, SSR3, CST3, ZNF266, CDC42, CD63, HIF1A, YWHAE, EIF3E, and PPIB. Independent RT-qPCR analyses were conducted confirming expression stability in another set of CL tissues from pregnancy and regression, with analyses performed for 3 groups of samples: (1) all samples, (2) samples from pregnancy alone, and (3) samples throughout the process of CL regression. Seven genes were found to be more stable in all states than 2 traditional reference genes (ACTB and GAPDH): RPS4X, COX4I1, PPIB, SSR3, RPL4, YWHAE, and CDC42. When CL tissues from pregnant animals alone were analyzed, CST3, HIF1A, and CD63 were also identified as more stable than ACTB and GAPDH. Identification of these new reference genes will aid in accurate normalization of RT-qPCR results, contributing to proper interpretation of gene expression relevant to luteal physiology. Furthermore, our analysis sheds light on the effects of luteolysis and pregnancy on the stability of gene expression in the bovine CL.

Honeybee wings hold antibiofouling and antimicrobial clues for improved applications in health care and industries.

Natural surfaces with remarkable properties and functionality have become the focus of intense research. Heretofore, the natural antimicrobial properties of insect wings have inspired research into their applications. The wings of cicadas, butterflies, dragonflies, and damselflies have evolved phenomenal anti-biofouling and antimicrobial properties. These wings are covered by periodic topography ranging from highly ordered hexagonal arrays of nanopillars to intricate "Christmas-tree" like structures with the ability to kill microbes by physically rupturing the cell membrane. In contrast, the topography of honeybee wings has received less attention. The role topography plays in antibiofouling, and antimicrobial activity of honeybee wings has never been investigated. Here, through antimicrobial and electron microscopy studies, we showed that pristine honeybee wings displayed no microbes on the wing surface. Also, the wings displayed antimicrobial properties that disrupt microbial cells and inhibit their growth. The antimicrobial activities of the wings were extremely effective at inhibiting the growth of Gram-negative bacterial cells when compared to Gram-positive bacterial cells. The fore wing was effective at inhibiting the growth of Gram-negative bacteria compared to Gram-positive samples. Electron microscopy revealed that the wings were studded with an array of rough, sharp, and pointed pillars that were distributed on both the dorsal and ventral sides, which enhanced anti-biofouling and antimicrobial effects. Our findings demonstrate the potential benefits of incorporating honeybee wings nanopatterns into the design of antibacterial nanomaterials which can be translated into countless applications in healthcare and industry.

Use of a wastewater recovery product (struvite) to enhance subtropical seagrass restoration.

Seagrasses are in decline worldwide, and their restoration is relatively expensive and unsuccessful compared to other coastal systems. Fertilization can improve seagrass growth in restoration but can also release nutrients and pollute the surrounding ecosystem. A slow-release fertilizer may reduce excessive nutrient discharge while still providing resources to the seagrass's rhizosphere. In this study, struvite (magnesium ammonium phosphate), a relatively insoluble, sustainable compound harvested in wastewater treatment plants, was compared to Osmocote™(14:14:14 Nitrogen: Phosphorus: Potassium, N:P:K), a popular polymer coated controlled release fertilizer commonly used in seagrass restoration. Two experiments compared the effectiveness of both fertilizers in a subtropical flow-through mesocosm setup. In the first experiment, single 0.5 mg of P per g dry weight (DW) doses of Osmocote™and struvite fertilizers were added to seagrass plots. Seagrass shoot counts were significantly higher in plots fertilized with struvite than both the Osmocote™and unfertilized controls (p< 0.0001). A significant difference in total P concentration was observed in porewater samples of Osmocote™vs struvite and controls (p< 0.0001), with struvite fertilized plots emitting more than controls (p ≤ 0.0001), but less than 2% of the total dissolved P (TDP) of Osmocote™fertilized plots (100+ mg/L versus x > 5 mg/L). A subsequent experiment, using smaller doses (0.01 and 0.025 mg of P per gram DW added), also found that the struvite treatments performed better than Osmocote™, with 16-114% more aboveground biomass (10-60% higher total biomass) while releasing less N and P. These results indicate the relatively rapid dissolution of Osmocote™may pose problems to restoration efforts, especially in concentrated doses and possibly leading to seagrass stress. In contrast, struvite may function as a slow-release fertilizer applicable in seagrass and other coastal restoration efforts.

Effect of route of administration of dinoprost tromethamine on plasma profiles of 13,14-dihydro-15-keto-prostaglandin F2α and progesterone in lactating Holstein cows.

Lutalyse HighCon (dinoprost tromethamine; Zoetis) has been approved for use both intramuscularly and subcutaneously in lactating dairy cows, although the effect of route of administration on circulating 13,14-dihydro-15-keto-prostaglandin F2α (PGFM), the metabolite of PGF2α, has not been evaluated. Multiparous, lactating Holstein cows were submitted to an Ovsynch protocol in which the last GnRH treatment (G2) was designated as d 0. Cows were fitted with indwelling jugular catheters on d 6 and administered 25 mg of dinoprost tromethamine (2 mL of Lutalyse HighCon) on d 7 either subcutaneously in the neck (SC; n = 6) or intramuscularly in the semitendinosus muscle (IM; n = 6). Blood samples were collected every 15 min after treatment for 1.75 h, then every 2 h for 48 h, and at 60 and 72 h, with the last time point corresponding to when cows would have received timed AI at 72 h within an Ovsynch protocol. Circulating PGFM concentrations were greater for SC than for IM cows from 15 to 90 min after treatment, which resulted in a greater area under the PGFM curve during the first 90 min after treatment (means ± SEM; 1,664 ± 129 pg·h/mL vs. 1,146 ± 177 pg·h/mL for SC vs. IM cows, respectively). This resulted in complete luteolysis in all but one cow in the SC treatment at 56 h, when GnRH would have been administered if dinoprost tromethamine had been administered as part of an Ovsynch protocol for timed AI. For cows that underwent complete luteal regression, circulating P4 did not differ between treatments at any time point. Thus, although SC cows had increased circulating PGFM 15 to 90 min after treatment, there was no difference in circulating P4 during induced luteolysis based on route of dinoprost tromethamine administration.

Short term, repeated exposure to rams during the transition into the breeding season improves the synchrony of mating in the breeding season.

The ram effect is widely used in Mediterranean breeds of sheep but its use in temperate genotypes is restricted by breed seasonality. However, ewes from these highly seasonal genotypes are sensitive to stimulation by rams close to the onset of the natural breeding season. In this study we developed a pre-mating protocol of repeated, short-term exposure to rams (fence-line contact or vasectomised rams) beginning during late anoestrus and continuing into the breeding season. We hypothesised that this pre-mating protocol would synchronise the distribution of mating of North of England Mule ewes during the breeding season above that observed in ewes isolated from rams prior to mating. Ram-exposed ewes were given contact with rams (Experiment 1: fence-line; FR, n=94 and Experiment 2: vasectomised rams; VR; n=103) for 24h on Days 0 (10 September), 17 and 34 of the experiment. Control ewes (Experiment 1; FC, n=98 and Experiment 2; VC; n=106) remained isolated from rams prior to mating. In Experiment 2, a subset of VR (n=35) and VC ewes (n=35) were blood sampled twice weekly to monitor their pre-mating progesterone profiles. At mating, harnessed entire rams were introduced, 17 or 16 days after the last ram exposure (Experiments 1 and 2) and raddle marks were recorded daily. The median time from ram introduction to mating was reduced in ewes given both fence-line and vasectomised ram contact (P<0.001), leading to a more compact distribution of mating and lambing (At least P<0.01). In the blood sampled VR ewes, there was a progressive decline in the number of days from ram exposure to the onset of dioestrus (at least P<0.05). This observation indicates that the cycles in VR ewes became increasingly synchronised over the pre-mating period, a pattern not evident in VC ewes. In conclusion, repeated, short-term exposure of ewes to rams during the transition into the breeding season is an effective method of synchronising the distribution of mating during the breeding season.

Prior exposure of maiden ewes to rams enhances their behavioural interactions with rams but is not a pre-requisite to their endocrine response to the ram effect.

In this study, we tested whether prior experience with rams would modify the behavioural and endocrine responses of maiden ewes to rams. During mid-anoestrus, sexually naïve, maiden ewes were exposed to rams for 7 days (ram experienced, RE; n=61) or isolated from rams (ram naïve, RN; n=63). All ewes were subsequently isolated from rams. In Experiment 1, RE (n=55) and RN (n=57) ewes were introduced to rams during late anoestrus. RE ewes had more total and positive interactions with rams than RN ewes (P<0.001). RE ewes showed more ram seeking behaviour and spent more time in proximity of rams than RN ewes (at least; P<0.05). In Experiment 2, RE (n=6) and RN (n=6) ewes were introduced to rams midway through a frequent blood sampling regime in late anoestrus. Ram introduction stimulated an increase in LH pulse frequency and basal LH in both RE and RN ewes (at least P<0.05). RE ewes had an increase in mean LH concentrations (P<0.01) that failed to reach significance in RN ewes (P<0.1). There was no significant effect of prior experience with rams on LH pulse frequency, amplitude or whether ewes had an LH surge. In conclusion, prior experience with rams is important in developing appropriate ewe-ram interactions but is not a pre-requisite to the endocrine response to the ram effect.

Effects of treatment with GnRH from 4 to 8 weeks of age on the attainment of sexual maturity in bull calves.

In bull calves an early transient increase in circulating concentrations of LH occurs between 6 and 20 weeks of age. This has been shown to influence reproductive development and performance later in life. In an attempt to hasten the onset of sexual maturity, bull calves (Hereford x Charolais) were treated (im) with 120 ng/kg of GnRH (n=6) twice every day from 4 to 8 weeks of age; control calves received saline (n=6). Injection of GnRH resulted in an LH pulse in all animals. GnRH treated bulls displayed more rapid testicular growth rates between 22 and 44 weeks of age. Sexual maturity (SC>or=28 cm) was achieved earlier in GnRH treated bulls compared to saline treated bulls (41.7+/-2.22 and 47.0+/-0.45 weeks of age, respectively) and this was confirmed by age of sexual maturity based on ejaculate characteristics (>50 million spermatozoa, >10% motility; 45.0+/-0.86 and 49.0+/-1.13 weeks of age for GnRH and control treated bull calves, respectively; P<0.05). We concluded that treatment with GnRH, twice daily, from 4 to 8 weeks of age, prior to the endogenous early increase in plasma LH concentrations, could increase in plasma LH concentrations, advance testicular development and reduce age at puberty in beef bull calves. This may provide the basis for a simple regimen to hasten sexual development in the bull calf.

The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season.

Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n=3) [corrected] and late luteal (LL; n=6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentration [corrected] of LH (at least P<0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency (P<0.01); EL, ML and LL ewes also had an increase in mean LH concentration (P<0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle.

Treating heifers with GnRH from 4 to 8 weeks of age advanced growth and the age at puberty.

In heifer calves, an early transient increase in circulating concentrations of LH is associated with early follicular development and is thought to regulate the timing of puberty. In an attempt to hasten the onset of sexual maturity, the early rise in LH concentration was advanced by injecting heifer calves with 120 ng/kg of GnRH (n=6) twice daily from 4 to 8 weeks of age; control calves received saline (n=6). Blood samples were collected every 15 min for 10h at 4, 8, 14, 20, 26, 32, 38, 44 and 50 weeks of age. Treatment with GnRH increased mean circulating concentrations of LH at 8 weeks of age (P<0.05), LH pulse frequency at 4 and 8 weeks of age (P<0.05), and reduced the mean age at puberty by 6 weeks (56.8+/-1.7 versus 62.8+/-2.4 weeks of age, for GnRH treated and control calves, respectively; P=0.04). Body weight gain was greater in GnRH-treated calves than control calves (P<0.05), and the rate of weight gain was shown to be a significant covariate within age at puberty. In conclusion, we suggest that the timing of the early rise in LH concentrations is a critical signal involved in the timing of puberty in heifers.

The effects of ram exposure during progestagen oestrus synchronisation and time of ram introduction post progestagen withdrawal on fertility in ewes.

Three experiments were undertaken to investigate the effect of a pre-mating ram exposure during progestagen synchronisation treatment on time of breeding, ovulation rate, embryo quality and fertility and any interaction with time of ram introduction for breeding post sponge withdrawal. Crossbred ewes in experiment 1a (n = 348), 1b (mule; n = 133) and 2 (n = 58) underwent a 12-14 days synchronisation protocol. Three days prior to sponge withdrawal ewes were divided into Control (ewes in continued isolation from rams) or +Ram (ram-exposed) groups. Rams were introduced to +Ram ewes and remained with ewes until sponge withdrawal. Ewes in experiments 1a and 2 received eCG at sponge withdrawal and were reintroduced to rams at either 36 or 48 h post sponge removal (PSR). In experiment 1b, ewes did not receive eCG and were reintroduced to rams at 24 h PSR. In experiments 1a and 1b time of breeding, date of lambing and litter size were recorded. In experiment 2, ewes were slaughtered 5 days post breeding, reproductive tracts flushed and corpora lutea, ova and embryos assessed. Fewer +Ram ewes were mated by 96 h PSR (P < 0.001) than Control ewes in experiment 1a but not when rams were introduced earlier in experiment 1b. In experiment 1a, ram introduction at 36 h PSR improved conception to first service compared to introduction at 48 h PSR (P < 0.01) in both +Ram and Control groups. In experiments 1a and 1b, +Ram ewes had reduced litter size caused by more single births (1a; P < 0.001, 1b; P < 0.01). In experiment 2, +Ram ewes had fewer corpora lutea than Control ewes (P < 0.001) but embryo quality was similar. However, more good embryos were produced when rams were introduced for breeding at 36 h compared to 48 h PSR (P < 0.001). We conclude that a pre-mating ram exposure during the synchronisation treatment reduced the number of ewes mated at and conceiving to the first service. This was partially overcome by introducing rams for breeding earlier (24 or 36 h compared to 48 h PSR) but the most dramatic decrease in fertility was due to a reduction in ovulation rate in the ram-exposed ewes.

Treatment of ovariectomized heifers with bovine follicular fluid specifically suppresses pituitary levels of FSH-beta mRNA.

To investigate the inhibin-induced suppression of FSH secretion by the anterior pituitary, chronically ovariectomized heifers (three per group) were treated for 56-58 h with either steroid-free bovine follicular fluid (bFF; 8 ml i.v. every 8 h) or 0.9% (w/v) NaCl (8 ml i.v. every 8 h). Blood was withdrawn at 8-h intervals for analysis of plasma concentrations of FSH and LH by radioimmunoassay. At the end of the treatment period, heifers were slaughtered and pituitary glands recovered for determination of gonadotrophin contents and levels of mRNA encoding FSH-beta, LH-beta, TSH-beta and common alpha glycoprotein hormone subunits using [32P]cDNA probes in total RNA dot and Northern blot assays. Treatment with bFF markedly suppressed plasma FSH by 85% (P less than 0.001 compared with pretreatment period), but did not affect plasma LH concentrations. Plasma FSH and LH concentrations did not vary significantly in the saline-injected control heifers. The level of FSH-beta subunit mRNA was reduced by 60% (P less than 0.001) in heifers treated with bFF, whereas no significant differences between control and bFF-treated heifers were observed in the levels of mRNA encoding LH-beta, TSH-beta or common alpha subunits. Treatments with bFF, however, did not affect pituitary content of either FSH or LH. These results support the conclusion that inhibin exerts its selective suppressive effect on the secretion of FSH by the bovine pituitary, at least in part, by directly inhibiting expression of the gene encoding the FSH-beta subunit.

Evidence that the bovine ovary secretes large amounts of monomeric inhibin alpha subunit and its isolation from bovine follicular fluid.

Analysis of bovine follicular fluid (FF) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with a sensitive immunoblotting procedure resolved several components that were immunoreactive with an antiserum directed against the n-terminus of the alpha subunit of human inhibin (hI alpha(1-32]. Under non-reducing conditions, three intensely stained bands having apparent Mr values of 116,000, 44,000 and 25,000 were present, whilst under reducing conditions only two intensely stained bands (Mr 43,000 and 21,000) were detected. The Mr 44,000 and 25,000 immunoreactive forms (non-reducing conditions) were also demonstrated in bovine utero-ovarian vein and peripheral venous plasma after subjecting samples (40 ml) to immunoaffinity concentration using Sepharose beads coupled to anti-hI alpha(1-32), SDS-PAGE and immunoblotting. The same approach revealed the presence of the smaller (Mr 25,000) form in bovine granulosa cell-conditioned culture medium (GCCM). Gel-permeation chromatography (Sephacryl S-200), immunoaffinity chromatography (Sepharose-anti-hI alpha(1-32] and reversed-phase high-performance liquid chromatography (RP-HPLC; C18 and C8 columns) were employed to isolate from bFF (30 ml, 19.5 g protein) 750 micrograms protein which appeared essentially homogeneous by RP-HPLC and SDS-PAGE and had an Mr of 25,000 (non-reducing conditions)/21,000 (reducing conditions), identical to that of the immunoreactive component of lowest Mr found in bovine FF, utero-ovarian vein plasma, peripheral plasma and GCCM. The isolated material was highly immunoreactive with antisera against both hI alpha(1-32) and purified Mr 32,000 bovine inhibin but was devoid of biological activity when tested in a rat pituitary cell inhibin bioassay. Amino-terminal analysis revealed an amino acid sequence (residues 1-14) identical to that reported elsewhere for the alpha subunit (Mr 20,000/21,000) of bovine inhibin. In conclusion, the present study has revealed that the bovine ovary secretes considerable quantities of monomeric inhibin alpha subunit. The unexpected presence of this material in peripheral blood is likely to hinder attempts to obtain physiologically relevant data on circulating levels of inhibin in cattle using conventional radioimmunoassays.

Development of a two-site immunoradiometric assay for dimeric inhibin using antibodies against chemically synthesized fragments of the alpha and beta subunit.

A two-site (liquid-phase) immunoradiometric assay (IRMA) for dimeric inhibin has been developed using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the alpha subunit and the C-terminal region (82-114) of the beta A subunit of Mr approximately 30,000 human inhibin. Highly-purified Mr 32,000 bovine inhibin (standard) gave a dilution curve parallel to those for bovine follicular fluid (bFF), human (h)FF and rat ovary extract. Whilst the assay detected both Mr 56,000 and 32,000 inhibin forms in bFF, little reaction with higher Mr forms was evident. Cross-reaction of 'free' inhibin subunit (Mr 25,000 form) and recombinant human activin A in the IRMA were minimal (less than 0.1 and less than 2% respectively). Although the detection limit of the IRMA (approximately 50 pg/tube) was similar to that of several reported radioimmunoassays (RIA), the IRMA was unable to detect dimeric inhibin in jugular or utero-ovarian vein plasma of heifers. Similarly, when assayed by IRMA, bFF, hFF and rat ovary extract contained 8-58 times less inhibin than was indicated by RIA. These observations are consistent with earlier evidence that the ovary secretes a substantial excess of 'free' inhibin alpha subunit and that this material reaches the peripheral circulation. Surprisingly, however, the inhibin contents of bFF, hFF and rat ovary extract determined by in vitro bioassay were 8-23 times greater than the corresponding IRMA values, being similar to those derived by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of crude and highly purified bovine inhibin (Mr 32,000) on plasma concentrations of FSH and LH in chronically ovariectomized prepubertal heifers.

Chronically ovariectomized prepubertal heifers were used for a comparison of the effects of highly purified bovine inhibin (Mr 32,000) and steroid-free bovine follicular fluid (bFF) on the secretion of FSH and LH. In view of the limited availability of highly purified inhibin, an initial study was undertaken to establish the optimal method for administration of bFF inhibin activity. In comparison with the FSH response to a single large i.v. bolus injection of bFF (50 ml; 3250 mg protein), a far more effective suppression of plasma FSH concentrations was achieved when considerably less bFF (6.3 ml; 410 mg protein) was administered gradually over an extended time-period (2 days) either as a continuous i.v. infusion or as a series of 2-hourly i.v. injections. Following a single i.v. bolus injection of bFF, immunoreactive inhibin was cleared rapidly from the circulation (half-life 51 +/- 8 (S.E.M.) min, n = 5), presumably accounting for its limited ability to suppress FSH secretion when administered in this manner. In a second experiment, treatment of ovariectomized heifers (three per group) with highly purified Mr 32,000 bovine inhibin at a dose rate of 15 micrograms/2 h for 2 days significantly (P less than 0.05) suppressed plasma FSH concentrations, which reached their minimum values (40% suppression) during day 2 of treatment. At a lower dose rate (5 micrograms/2 h), inhibin did not significantly affect plasma FSH levels. Administration of bFF was also associated with a dose-dependent suppression of FSH secretion. For each of three dose rates tested (three heifers per group), plasma FSH concentrations were maximally suppressed during day 2 of treatment (65 mg/2 h, 86% suppression, P less than 0.001; 21.7 mg/2 h, 66% suppression, P less than 0.001; 7.2 mg/2 h, 15% suppression, P greater than 0.05). Neither highly purified inhibin nor bFF significantly affected mean plasma LH concentrations, LH pulse frequency or LH pulse amplitude. Thus we have shown for the first time that highly purified Mr 32,000 bovine inhibin does possess in-vivo biological activity in cattle, promoting a selective suppression of plasma FSH concentrations qualitatively similar to that evoked by steroid-free bFF.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin alpha subunit: effects on gonadotrophin concentrations and ovulation rate in sheep.

Two experiments were conducted to explore the effectiveness of synthetic peptide-based vaccines for active and passive autoimmunization of sheep against inhibin. In the first experiment, adult Romney ewes (n = 20) were actively immunized against a synthetically produced peptide that corresponded to the N-terminus of the alpha-subunit of bovine inhibin (bI alpha(1-29)-Tyr30). This peptide was conjugated to tuberculin purified protein derivative (PPD) to increase its antigenic properties. Control groups comprised non-immunized (n = 10) and PPD-immunized (n = 10) ewes. Primary immunization (400 micrograms conjugate/ewe) was followed by two booster immunizations (200 micrograms conjugate/ewe), given 5 and 8 weeks later. Following synchronization of oestrus using progestagen sponges, ovulation rates were assessed by laparoscopy. Weekly blood samples were taken throughout the experiment. All inhibin-immunized ewes produced antibodies which bound 125I-labelled bovine inhibin (Mr 32,000), and ovulation rate in inhibin-immunized ewes (2.15 +/- 0.22; mean +/- S.E.M.) was significantly (P less than 0.01) greater than in both non-immunized (0.90 +/- 0.23) and PPD-immunized (1.20 +/- 0.13) control groups. Immunization against the peptide, but not against PPD alone, resulted in a modest rise in plasma FSH, with mean levels after the second boost being significantly (P less than 0.025) higher (22%) than those before immunization. Moreover, when blood samples were taken (2-h intervals) from randomly selected groups of control (n = 7) and inhibin-immunized (n = 7) ewes for an 84-h period following withdrawal of progestagen sponges, the mean plasma concentration of FSH during the 48 h immediately before the preovulatory LH surge was 37% greater (P less than 0.025) in immunized than in control animals. However, more frequent blood sampling (every 15 min for 12 h) during follicular and mid-luteal phases of the oestrous cycle revealed no significant differences between treatment groups in mean plasma concentrations of FSH. In addition, neither mean concentrations of LH nor the frequency and amplitude of LH episodes differed between immunized and control ewes. However, the mean response of LH to a 2 micrograms bolus of gonadotrophin-releasing hormone, given during the luteal phase, was significantly (P less than 0.05) less in immunized than in control ewes. These findings indicate that active immunization of Romney ewes against a synthetic fragment of inhibin can promote a controlled increase in ovulation rate, but this response cannot be unequivocally related to an increase in plasma levels of FSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of active immunization of heifers against inhibin on plasma FSH concentrations, ovarian follicular development and ovulation rate.

To study the effects of immunoneutralization of endogenous inhibin on gonadotrophin secretion and ovarian function, prepubertal heifers (n = 6) were actively immunized against a synthetic peptide replica of the N-terminal sequence of bovine inhibin alpha subunit (bI alpha(1-29)Tyr30) coupled to ovalbumin. In contrast to ovalbumin-immunized controls (n = 6), bI alpha(1-29)Tyr30-immunized heifers had detectable inhibin antibody titres (% binding to 125I-labelled bovine inhibin at 1:2000 dilution of plasma) of 17 +/- 3% (S.E.M.) at puberty, rising to 31 +/- 5% by the end of the study period 7 months later. Neither age (immunized: 295 +/- 8 days; controls: 300 +/- 5 days) nor body weight (immunized: 254 +/- 13 kg; controls 251 +/- 9 kg) at onset of puberty differed between groups. Although the difference did not reach statistical significance, mean plasma FSH concentrations recorded in inhibin-immunized heifers remained 35-40% higher than in controls throughout the 12-week period leading up to puberty (P = 0.14) and during nine successive oestrous cycles studied after puberty (P = 0.10). Plasma LH concentrations did not differ between groups at any time during the study. Inhibin immunization had no effect on oestrous cycle length (immunized: 19.8 +/- 0.5 days; controls: 19.9 +/- 0.5 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in inhibin-A (alpha-beta A dimer) and total alpha inhibin in the peripheral circulation and ovaries of rats after gonadotrophin-induced follicular development and during the normal oestrous cycle.

Recent modifications to a previously reported two-site IRMA have permitted the measurement of serum/plasma concentrations and ovarian contents of inhibin-A (alpha-beta A dimer) in pregnant mare serum gonadotrophin (PMSG)-treated immature female rats and adult rats throughout the 4-day oestrous cycle. For comparison, total alpha inhibin levels were also measured by alpha subunit-directed inhibin RIA and found to be at least tenfold higher (relative to the same 32 kDa bovine inhibin standard used to calibrate both assays). In immature female rats, serum levels of inhibin-A dimer and total alpha inhibin increased within 3 h of PMSG injection and rose in parallel over the next 48 h to values four- to fivefold higher than pretreatment levels. Ovariectomy led to a rapid and parallel fall in both inhibin-A dimer and total alpha inhibin; initial half-lives (+/- 95% confidence intervals) were 22 +/- 4 and 20 +/- 5 min respectively. In adult rats, marked fluctuations in plasma concentrations and ovarian contents of inhibin-A dimer and total alpha inhibin occurred during the 4-day oestrous cycle, most notably between the morning of pro-oestrus and the morning of oestrus. Plasma levels of inhibin-A dimer and total alpha inhibin peaked on the afternoon of pro-oestrus just before the preovulatory gonadotrophin surge. After ovulation, both inhibin-A dimer and total alpha inhibin fell abruptly (two- to threefold by 0200 h on oestrus; P < 0.001), while FSH showed a secondary rise which peaked at 0700 h on oestrus. Although IRMA- and RIA-derived inhibin values generally followed a similar pattern across the 4-day cycle (plasma: r = 0.52, P < 0.001; ovary: r = 0.41, P < 0.001), a transient rise in plasma and ovarian inhibin-A dimer was detected at 0700 h on oestrus (P < 0.01) which was unaccompanied by a rise in total alpha inhibin. This rise in plasma inhibin-A dimer was probably responsible for terminating the post-ovulatory FSH surge since FSH levels declined steadily over the next 15 h. Overall, plasma inhibin-A dimer and FSH concentrations across the whole cycle were negatively correlated (r = -0.22, P < 0.01) whereas no correlation existed between total alpha inhibin and FSH (r = -0.11, P = 0.12).

The ability of steroid-free bovine follicular fluid to suppress FSH secretion and delay ovulation persists in heifers actively immunized against inhibin.

Previous reports indicate that administration of steroid-free bovine follicular fluid (bFF) to intact heifers suppresses plasma FSH levels and delays the timing of ovulation. In addition, cessation of bFF treatment is associated with a rebound hypersecretion of FSH. To test the hypothesis that these effects of bFF are mediated by inhibin, we have compared the responses to bFF treatment in heifers actively immunized against the N-terminal sequence of inhibin alpha subunit (bI alpha(1-29)Tyr30-ovalbumin) with those in ovalbumin-immunized controls. Oestrous cycles were synchronized, and inhibin-immune (n = 10) and control (n = 10) heifers were subdivided into two groups which received either 5 ml bFF (n = 5) or 5 ml bovine serum (n = 5) every 4 h for a 60 h period starting 8 h before prostaglandin (PG)-induced luteolysis. Blood was withdrawn every 4 h for 10 days starting 30 h before luteolysis and ovaries were examined daily by ultrasonography. Overall, mean ovulation rate in bI alpha(1-29)Tyr30-immunized heifers was 44% higher (P < 0.02) than in controls. Inhibin antibody titres tested in bI alpha(1-29)Tyr30-immunized heifers before (19 +/- 3%), during (19 +/- 3%) and after (20 +/- 3%) bFF treatment did not differ. In the pretreatment period (i.e. mid-luteal phase), plasma FSH levels were 32% (P < 0.05) higher in inhibin-immunized than in control heifers. During administration of bFF to control heifers, plasma FSH was suppressed to a level 40% lower than in serum-treated heifers (P < 0.02). Unexpectedly, bFF suppressed FSH to a similar extent in inhibin-immunized heifers (37% lower than in the serum-treated group; P < 0.025). Similarly, a post-bFF rebound hypersecretion of FSH was observed in both control (P < 0.05) and inhibin-immunized (P < 0.05) heifers, although the FSH rebound lasted about 24 h longer in the latter group (P < 0.001). The timing of the preovulatory LH surge in control (86 +/- 7 h post-PG) and immunized (81 +/- 6 h post-PG) groups treated with serum was similar as was the timing of the preovulatory rise in plasma oestradiol and the subsequent rise in plasma progesterone. However, bFF treatment delayed (P < 0.001) the preovulatory surges of LH and oestradiol and the subsequent rise in plasma progesterone to a similar extent (> 4 days) in both control and inhibin-immunized groups.(ABSTRACT TRUNCATED AT 400 WORDS)