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.

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.

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.

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.

Outcome of reconstruction for duplication of the thumb in adults aged over 40.

Congenital duplication of the thumb is usually reconstructed by 18 months of age. This paper reports satisfactory outcome following reconstruction of two Wassel Type IV duplicate thumbs in adults aged over 40. Both achieved a good outcome based on Tada's criteria and a subjective Visual analogue score 8.

Ram novelty and the duration of ram exposure affects the distribution of mating in ewes exposed to rams during the transition into the breeding season.

This study compared the affect of short-term and continuous exposure to rams during the transition between anoestrus and the breeding season on the distribution of mating and subsequent lambing. Further, within ewes continuously exposed to rams we investigated the effect of replacing these rams every 17 days with 'novel' rams. During August (late anoestrus, Northern Hemisphere), multiparous, North of England mule ewes were allocated to one of four groups: SVR ewes were exposed to vasectomised rams for 24h on Day 0 (short term; n=109), RVR ewes were exposed to vasectomised rams for 24h on Days 0, 17 and 34 (short term; n=113); PVR ewes were exposed to vasectomised rams on Day 0 and remained with the same rams for the duration of the pre-mating period (continuous; n=104); NVR ewes were continuously exposed to vasectomised rams from Day 0 with the rams replaced with 'novel' rams every 17 days (continuous; n=113). Blood samples were collected from a subset of ewes (n=22 per group) to monitor progesterone. On Day 50, harnessed, entire rams were introduced for mating and raddle marks recorded daily for the first 17 days. The median date of mating occurred 1 day earlier in NVR ewes than PVR ewes (P<0.05). A synchrony score calculated from the blood sampled ewes showed that the distribution of mating was more synchronised in PVR and NVR ewes than SVR and RVR ewes (P<0.001). PVR and NVR ewes had an earlier onset of cyclic activity than RVR ewes (P<0.01). However, only NVR ewes differed from SVR ewes in this variable (P<0.05). Within ewes lambing to first service, the median date of lambing of PVR, NVR and SVR ewes occurred at least 2 days earlier than RVR ewes (at least P<0.05). Further, PVR and NVR ewes had a more compact distribution of lambing than SVR and RVR ewes (P<0.05) and lambing was more compact in NVR ewes than PVR ewes (P<0.05). In conclusion, ewes in continuous contact with rams prior to mating had a more synchronised distribution of mating and lambing than ewes given only short-term exposure to rams. This distribution of mating in continuous ram exposed ewes can be further enhanced by periodic exposure to novel rams.

Neonatal lamb vigour is improved by feeding docosahexaenoic acid in the form of algal biomass during late gestation.

To determine whether feeding a sustainable, algal source of docosahexaenoic acid (DHA) to sheep during late pregnancy would improve neonatal lamb vigour, 48 English mule ewes, of known conception date, were divided into four treatment groups. For the last 9 weeks of gestation, ewes received one of two dietary supplements: either a DHA-rich algal biomass providing 12 g DHA/ewe per day, or a control supplement based on vegetable oil. The four dietary treatment groups (n = 12) were: control supplement for the duration of the trial (C), DHA supplement from 9 to 6 weeks before parturition (3 week), DHA supplement from 9 to 3 weeks before parturition (6 week) and DHA supplement for the duration of the trial (9 week). Dietary supplements were fed alongside grass silage and commercial concentrate. There was a tendency for gestation length to be extended with increased duration of DHA supplementation (P = 0.08). After parturition, the concentrations of eicosapentaenoic acid (EPA) and DHA in ewe and lamb plasma and colostrum were elevated in line with increased periods of DHA supplementation. Lambs from the 6-week and 9-week groups stood significantly sooner after birth than lambs from the C group (P < 0.05). These data show that neonatal vigour may be improved by the supplementation of maternal diets with DHA-rich algal biomass and that this beneficial effect depends upon the timing and/or duration of DHA allocation.

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.

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.

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.

Safety, pharmacokinetics, and efficacy of (+/-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine with efavirenz and stavudine in antiretroviral-naïve human immunodeficiency virus-infected patients.

Racivir [RCV; (+/-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine], a 50:50 racemic mixture of the two beta nucleoside enantiomers, is currently in development for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. RCV was administered once a day orally for 14 days at doses of 200, 400, or 600 mg in combination with stavudine and efavirenz to HIV-1-infected treatment-naïve male volunteers in a phase Ib/IIa study. Six volunteers at each dose were monitored for a total of 35 days for tolerance, pharmacokinetics, and plasma HIV RNA levels. RCV in combination with stavudine and efavirenz was well tolerated at all doses tested. Pharmacokinetic parameters were dose proportional, and the maximum concentration of drug in serum at all doses exceeded the 90% effective concentration for wild-type HIV-1. Viral loads dropped as expected in all dosage groups, with mean reductions from 1.13 to 1.42 log10 by day 4 and 2.02 to 2.43 log10 by day 14. HIV RNA levels remained suppressed for more than 2 weeks in the absence of any additional therapy, with mean viral loads ranging from 2.1 to 2.6 log10 below baseline through day 28. By day 35, HIV RNA levels began to increase but still remained >1 log10 below baseline levels.

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.

Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes.

Two experiments were conducted to examine the effects of ram exposure during the breeding season, in combination with progestagen treatment on estrus synchronization, fertility the LH surge and ovulation in ewes. Experiment 1 was subdivided into experiments 1a and 1b. In all experiments cross-bred ewes were treated with an intravaginal sponge for 12-14 days and three days before sponge withdrawal ewes were divided into control (no further treatment; n=191, 103 and 50 for experiments 1a, 1b and 2, respectively) or ram exposed (three mature rams per 50 ewes were introduced; +Ram; n=187, 99 and 49 for experiments 1a, 1b and 2, respectively). At sponge withdrawal ewes in Experiments 1a and 2 received 500 IU eCG and rams were removed from all the +Ram groups. In Experiments 1a and 1b, raddled, entire rams were introduced to ewes 48 h after sponge withdrawal. The timing of mating was recorded and ewes were maintained until lambing. In Experiment 2, estrus behavior was determined every 4 h and the time of the LH surge and ovulation were determined from a subset of 10 ewes per group. In Experiment 1a, less +Ram ewes were bred by 48 h after ram introduction (control 98% versus +Ram 89%, P<0.001) and in Experiments 1a and 1b 14% fewer (P<0.05) of the ewes bred in the first 3 h after ram introduction lambed to that service. In Experiment 1a, ram exposed ewes had a lower litter size than control ewes (1.93+/-0.06 versus 1.70+/-0.06 lambs per ewe; P<0.05). In Experiment 2, rams advanced (P<0.05) estrus, the LH surge and ovulation by 2-6 h compared with control ewes. We speculate that exposure of ewes to rams increased LH secretion and that this in turn increased follicle development and the production of oestradiol that led to a more rapid onset of estrus, the LH surge and ovulation compared to control ewes. Unexpectedly, ewes that were bred had lower fertility in the +Ram groups than control groups.

Effects of medroxyprogesterone acetate (MAP) on ovarian antral follicle development, gonadotrophin secretion and response to ovulation induction with gonadotrophin-releasing hormone (GnRH) in seasonally anoestrous ewes.

When ovulation is induced with gonadotrophin-releasing hormone (GnRH) in anoestrous ewes, a proportion of animals fail to form normal (full-lifespan) corpora lutea (CL). Progesterone treatment before GnRH prevents luteal inadequacy. It remains uncertain whether a similar effect, achieved with medroxyprogesterone acetate (MAP) from intravaginal sponges, is mediated by influences on growing ovarian follicles and/or secretion of gonadotrophic hormones, before and after GnRH treatment. Two experiments were performed, on 13 and 11 anoestrous Western white-faced ewes, respectively. Seven and six ewes, respectively, received MAP-containing sponges (60 mg) for 14 days; the remaining ewes served as untreated controls. To test the effect of timing of GnRH administration after pre-treatment with MAP-releasing sponges, GnRH injections (250 ng every 2h for 24h followed by a bolus injection of 125 microg of GnRH i.v.) were given either immediately (Experiment 1) or 24h after sponge removal in the treated ewes (Experiment 2). Ovarian follicular dynamics (follicles reaching >or=5mm in size) and development of luteal structures were monitored using transrectal ultrasonography. In Experiment 1, the mean ovulation rate (0.7+/-0.3 and 1.0+/-0.4) and proportion of ovulating ewes (57 and 67%, respectively) did not vary (P>0.05) between MAP-treated and control ewes. Normal (full-lifespan) CL were detected in 29% of treated and 67% of control ewes (P>0.05). In Experiment 2, the mean ovulation rate (2.3+/-0.2 and 1.2+/-0.6; P<0.05) and percentage of ewes with normal (full-lifespan) CL (100 and 40%, respectively; P<0.10) were greater in the treated compared to control ewes. In Experiment 1, the mean peak concentration of the GnRH-induced LH surge was lower (P<0.05) in MAP-treated than in control ewes. There were no significant differences between MAP-treated and control ewes in the characteristics of follicular waves, mean daily serum FSH concentrations, and secretory parameters of LH/FSH, based on intensive blood sampling conducted 1 day before sponging and 1 day before sponge removal. It is concluded that treatment with MAP has no effect on the tonic secretion of LH/FSH or follicular wave development in anoestrous ewes. However, the GnRH-stimulated LH discharge was attenuated in the ewes that received MAP-impregnated sponges for 14 days and were treated with GnRH immediately after sponge withdrawal. Ovulatory response and CL formation were increased when GnRH was administered 24 h after sponge removal.

Ovarian responses in gonadotrophin-releasing hormone-treated anoestrous ewes: follicular and endocrine correlates with luteal outcome.

The relationships between the development of antral follicles (growing from 3 to > or = 5 mm diameter), hormone secretion (luteinizing hormone (LH), follicle-stimlating hormone (FSH), oestradiol and progesterone), ovulation and the formation of luteal structures in response to gonadotrophin-releasing hormone (GnRH) were examined in 24 anoestrous Western White Face ewes (May-July). Ewes were monitored by transrectal ovarian ultrasonography for 34 days, commencing 15 days before the administration of GnRH. Following treatment with GnRH, 83% (20/24) of ewes ovulated. Twenty-five per cent of all ewes (6/24) subsequently had normal (full-life span) corpora lutea (CL), 37% (9/24) had inadequate CL, 17% (4/24) had both normal and inadequate CL, 17% (including three of four anovular ewes and one ewe with inadequate CL) formed luteinized follicles and only 4% (1/24) did not ovulate or produce any luteal structure. None of the variables of follicular growth (follicles reaching > or = 5 mm diameter) differed between follicles that either ovulated or failed to ovulate and there was no evident correlation between the age or stage of development of ovulatory sized antral follicles and the type of luteal structure formed, except for luteinized unovulated follicles; these follicles all emerged within 3 days of GnRH injection. Mean serum concentrations of FSH and oestradiol before treatment did not differ (P>0.05) between ewes with different ovarian responses, but peaks of fluctuations in serum concentrations of FSH in daily samples were higher in ewes that produced normal CL compared with ewes with inadequate CL. After GnRH treatment, oestradiol secretion was higher in ewes that formed luteinized unovulated follicles than in all ewes with inadequate CL (P<0.05). The peak concentration of the GnRH-induced LH surge was higher and the interval from GnRH to peak LH discharge was shorter in ewes with inadequate CL compared with ewes that had normal CL after ovulation (P<0.05). In conclusion, ovulatory sized antral follicles at a similar stage of their life span can give rise to either normal or inadequate CL and a proportion of these follicles do not ovulate in response to GnRH in seasonally anoestrous ewes. This suggests differences in ovarian follicular responsiveness to gonadotrophic stimuli. Both the amplitude of episodic elevations in daily serum FSH concentrations and the characteristics of the pre-ovulatory LH surge may be important for luteogenesis following ovulation.