Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors.

In the first of two experiments investigating the effect of dietary urea on the survival and metabolism of ovine embryos, 30 Border Leicester x Scottish Blackface ewes received a maintenance diet (milled hay, molasses, minerals, vitamins) with no urea (control, C; n = 10) or with added urea at 15 g (low urea, LU; n = 10) or 30 g (high urea, HU; n = 10) kg-1 feed for a 12 week period. The degraded nitrogen (N) status relative to estimated rumen microbial N requirements was -2, +9 and +20 g per day, respectively. One week after allocation to diets, progesterone priming (12 days) commenced. Ewes received 800 IU of equine chorionic gonadotrophin at progesterone withdrawal, were inseminated 52 h later (Day 0) and embryos were collected from five ewes per group at Day 4 and from five ewes at Day 11. If available, one embryo was returned to each ewe; the rest were cultured in vitro. There was no effect of treatment on progesterone, luteinizing hormone (LH), or time of oestrus onset C, LU and HU plasma urea (P < 0.001) and ammonia levels (C vs. HU, P < 0.01; LU vs. HU, P < 0.05) differed. Day 4 HU embryos were retarded relative to C and LU embryos. After 3 days of culture, 70%, 66% and 0% of C, LU and HU embryos, respectively, were viable. Mid-term pregnancy rates following transfer were 63%, 43% and 33%. Only one HU lamb (male) was born following embryo transfer, its birthweight (10.1 kg) exceeded that of its C (n = 3; 7.0, 7.0, 7.5 kg) and LU (n = 2; 7.3, 8.2 kg) counterparts (P < 0.025). In the second experiment, C2 (2.5 g urea kg-1; n = 5) and HU2 (30 g kg-1; n = 7) diets which provided similar intakes of degraded N relative to microbial requirements as those for C and HU ewes in Experiment 1 were fed to Border Leicester x Scottish Blackface ewes superovulated with 16 mg of porcine follicle-stimulating hormone. Urea and ammonia levels in utero-oviductal samples were elevated in HU2 ewes (P < 0.05). At collection (Day 3), HU2 embryos used more glucose (P < 0.01) and, following culture, some exhibited up to a 2.8-fold increase in metabolism. In conclusion, excess rumen degradable N in ewe diets elevates urea and ammonia in plasma and in utero, with an associated increase in embryo mortality. Nevertheless, metabolism appears to be up-regulated in some embryos and, among those that survive, fetal growth appears to be enhanced.

The effect of two different levels of progesterone priming on the response of ewes to superovulation.

The use of either 1 or 3 controlled internal drug release (CIDR) devices for progesterone priming in ewes (n=11) superovulated with 1500 IU pregnant mare serum gonadotrophin (PMSG) at 28 hours prior to CIDR device withdrawal was investigated in relation to the stages of development and viability of the ova produced. Progesterone levels in the ewes (n=6) treated with 3 CIDR devices were significantly higher (P<0.01) during the 11 days of insertion than in those (n=5) treated with 1 CIDR device (7.3 vs 3.3 ng/ml) over the same period. However, following superovulation, the mean (+/-SEM) ovulation rates were similar for both groups (8.2 +/- 1.7 vs 10.2 +/- 1.5). The number of ova (M+/-SEM) recovered by laparoscopy 5 days after insemination was 4.2 +/- 1.0 for ewes treated with 3 CIDR devices and 7.0 +/- 1.1 for those treated with 1 CIDR device (P<0.10). The respective ovum recovery rates (M+/-SEM) were 55+/-9.8 and 74+/-13.2%. There was no effect of progesterone concentration in the priming phase on either the stages of development of the recovered ova or on their ability to develop during in vitro culture. It was concluded, therefore, that progesterone concentrations within the range 3.3 +/- 0.1 to 7.3 +/- 0.3 ng/ml during the priming phase and 2.4 +/- 0.3 to 6.5 +/- 0.2 ng/ml at the time of PMSG administration did not affect the ovulation rate or the viability of ova recovered from superovulated ewes.

Melatonin treatment of embryo donor and recipient ewes during anestrus affects their endocrine status, but not ovulation rate, embryo survival or pregnancy.

Thirty-two Border Leicester x Scottish Blackface ewes that lambed in March were individually penned with their lambs from April 16th and given daily an oral dose of 3 mg melatonin at 1500 h (Group M). A further 32 acted as controls (Group C). Within each group half were used as embryo donors (Group D) following superovulation and half received embryos (Group R) following an induced estrus. Prior to weaning on 21 May ewes received ad libitum a complete diet providing 9 megajoules (MJ) of metabolizable energy and 125 g/kg crude protein. Thereafter each received 1.6 kg of the diet daily. In early June each ewe received an intravaginal device (300 mg progesterone) inserted for 12 d. Donors were superovulated with 4 i.m. injections of porcine FSH 12 h apart, commencing 24 h before progesterone withdrawal. Ovulation in recipients was induced with 800 IU PMSG injected i.m. at progesterone removal. Donor ewes were inseminated 52 h after progesterone withdrawal. Embryos were collected 4 d later and transferred to recipients. Melatonin suppressed plasma prolactin (P < 0.001) and advanced estrus (P < 0.05) and timing of the LH peak (P < 0.05). These events also occurred earlier in donors than in recipients (P < 0.01). Mean (+/- SEM) ovulation rates for melatonin-treated and control donors were 5.5 +/- 0.71 and 4.7 +/- 0.66, respectively (NS). Corresponding recipient values were 3.3 +/- 0.40 and 3.4 +/- 0.39 (NS). Mean (+/- SEM) embryo yields were 2.9 +/- 0.64 and 2.6 +/- 0.73 for melatonin-treated (n = 15) and control (n = 16) donors, respectively, and for the 12 ewes per treatment that supplied embryos, corresponding numbers classified as viable were 2.7 +/- 0.47 and 2.3 +/- 0.61 (NS). Following transfer, 57% of embryos developed to lambs when both donor and recipient received melatonin, 86% when only the donor received melatonin, 91% when only the recipient received melatonin, and 67% when neither received melatonin (NS). Thus, embryo survival following transfer was not improved by treating recipients with melatonin. Gestation length and lamb birthweights were unaffected by melatonin. Unlike nonpregnant control ewes, melatonin-treated recipients that failed to remain pregnant sustained estrous cyclicity following embryo transfer.

The effect of time of intrauterine insemination on the development and viability of embryos collected from superovulated ewes.

Eighteen Border Leicester x Scottish Blackface ewes, primed with 300 mg progesterone (12 d) and superovulated with decreasing doses (6, 5, 3 and 2 mg) of porcine FSH, were inseminated with fresh semen, using laparoscopic intrauterine procedures at 48 (Group E) or 60 h (Group L) after exogenous progesterone removal. Five days after insemination, embryos were collected and classified on the basis of their morphological development. During the subsequent 3 d of in vitro culture (38.5 degrees C; 5% CO2) the embryos were evaluated at 24-h intervals. After 72 h, the embryos were individually fixed (24 h) and stained with aceto-orcein and the nuclei were then counted to provide an objective index of cell proliferation and development. Mean (+/-SEM) ovulation rates for the 2 groups (9.2+/-1.5 and 7.1+/-1.2, respectively) and the corresponding percentages (53 vs 59) of embryos collected by laparoscopy were unaffected by insemination time. All donors yielded fertilized ova, but whereas all Group-E donors yielded 1 or more viable embryos (i.e., >32 cells), only 5 Group-L ewes yielded viable embryos (P<0.10). At collection, the percentages of embryos at the morula stage of development were 98 (Group E n = 44) and 39 (Group L n = 38; P<0.001). Few of the remaining ova (Group E = 0% Group L = 8%) were at the 1-cell stage of development when collected, indicating that retarded development post fertilization, not fertilization failure, was the principal consequence of delayed insemination. The percentages of embryos that continued to develop during in vitro culture were 91 and 37 for Groups E and L, respectively (P<0.001), and all of these reached the blastocyst stage. Of these blastocysts, 75 and 50% in Groups E and L hatched in vitro (P<0.10), with mean (+/-SEM) nuclei counts of 148+/-22.7 and 76+/-13.8 (P<0.02), respectively. In conclusion, while delayed intrauterine insemination did not affect the efficiency of ovum collection, it caused a major reduction in the yield of embryos that were capable of developing during in vitro culture. However, fertilization failure accounted for only 13% of the loss in viability following late insemination.

Laparoscopy for intrauterine insemination and embryo recovery in superovulated ewes at a commercial embryo transfer unit.

Of 111 variable age, pedigree ewes subjected to a range of superovulatory regimens and then submitted to embryo recovery by laparoscopy, nine had adhesions corresponding to a mid-line laparotomy (presumably from a previous attempt to recover embryos) and could not have their embryos recovered by the laparoscopic technique. Of the remainder, 27 ewes (26.5%) had less than three ovulations or had prematurely regressing corpora lutea at the selected time for embryo recovery (Days 5 to 6 following insemination), and no attempt was made to recover embryos from them. For the 75 ewes subjected to laparoscopic ovum recovery following laparoscopic intrauterine insemination, the average number of ovulations (+/- SEM) was 7.9 +/- 0.6; the average ovum recovery (mean of values for each ewe) was 51.7% +/- 3.5; and the percentage of recovered ova that were fertilized was 87.3%. For a further nine 3-yr-old crossbred ewes the mean values for ovulation and ovum recovery were 7.6 +/- 1.2 and 70.1 +/- 7.7, and were not significantly different for the two insemination methods used (laparoscopic intrauterine vs cervical). In general, ovulation rates for ewes given pregnant mare serum gonadotrophin (PMSG) tended to be lower (5.2 +/- 0.7) than for those given porcine follicle stimulating hormone (pFSH, 7.7 +/- 0.8) or human menopausal gonadotrophin (hMG, 7.7 +/- 2.3). Ova recovery rates were similar on Days 5 and 6 (Day 0 = insemination), and were not affected by method of insemination (laparoscopic intrauterine vs cervical).

Behavioural responses of lambs of three ages in the first three hours after three methods of castration and tail docking.

The behavioural responses of groups of seven lambs were compared with control groups after castration and tail docking by rubber rings, application of a Burdizzo clamp in addition to a rubber ring and after surgical castration at five, 21 and 42 days. All methods at all ages produced changes in behaviour which were interpreted as indicative of considerable pain. The rubber ring groups showed most changes in behaviour at all ages. The rubber ring with Burdizzo groups showed least changes and some lambs in these groups showed much less response than others. The surgical groups showed some behavioural responses which were different, both qualitatively and quantitatively to those in other groups. It is concluded that indices used for recognition and assessment of acute pain received conditional support, that modification of the rubber ring with Burdizzo may provide the least painful method without local anaesthesia and that age had little effect on the responses.

Changes in plasma cortisol concentration in lambs of three ages after three methods of castration and tail docking.

Lambs were handled only or castrated and tail docked at five, 21 and 42 days of age by either surgery, rubber ring or rubber ring and Burdizzo. Plasma cortisol was measured in blood samples taken before and at 12, 24, 36, 48, 60, 72, 84, 96, 138 and 180 minutes after castration and docking. Pre-treatment and peak cortisol values were highest in five-day-old lambs. The peak cortisol values, at each age, were similar for surgery and rubber ring groups. However, the peak occurred earlier after surgery and rubber ring Burdizzo than after rubber ring only treatment. The cortisol peak was 28 nmol litre-1 lower after rubber ring Burdizzo than surgery or rubber ring only. Plasma cortisol returned to pretreatment values within 84 minutes after rubber ring Burdizzo, 96 to 138 minutes after rubber ring only but not within 180 minutes after surgery. The changes in plasma cortisol together with the changes in behaviour suggest that the rubber ring Burdizzo method of castration and docking of lambs at all ages, was probably the least painful of the methods tested.

Effect of different methods of castration on behaviour and plasma cortisol in calves of three ages.

Calves of six, 21 and 42 days were either handled (controls) or castrated by Burdizzo, by surgical or by rubber ring techniques and their behaviour and plasma cortisol monitored for three hours after treatment. The rubber ring caused significant increases in active behaviour and abnormal postures for two hours compared with handled or castrated by Burdizzo and surgical techniques, and surgical techniques resulted in a significant increase in abnormal standing, particularly in the first 30 minutes. Abnormal behaviour and posture were recorded less frequently in six-day-old calves. The mean cortisol peaks for six-, 21- and 42-day-old calves were, respectively, 36, 31 and 23 nmol litre-1 in the handled groups; 60, 43 and 48 nmol litre-1 in the rubber ring groups; 77, 50 and 62 nmol litre-1 in the Burdizzo groups; and 106, 63 and 107 nmol litre-1 in the surgical groups. The greatest cortisol response occurred in 42-day-old surgically treated calves and the shortest response after Burdizzo castration. These responses suggest that irrespective of age, all methods of castration studied caused acute pain. Burdizzo castration appeared to produce the least pain, particularly in younger calves.

Effects of castration on behaviour and plasma cortisol concentrations in young lambs, kids and calves.

Behavioural and cortisol responses to the husbandry practice of castration with tight rubber rings were investigated in lambs and kids one day after birth and in hand reared calves aged one to seven days. There were three treatments: control handling and blood sampling, castration and, in lambs and kids only, intravenous adrenocorticotrophin injection (ACTH). The integrated cortisol responses (area under the cortisol curve) in lambs and kids were least in control, intermediate in castrated and greatest in ACTH animals. No cortisol responses were detected in control or castrated calves. The incidences of behaviour used to assess the intensity of distress apparently experienced in the different species corresponded generally with the magnitudes of the cortisol responses. Behavioural and cortisol responses together suggested that the distress caused by castration was greatest in lambs, intermediate in kids and least but not necessarily absent in hand reared calves.

Comparison of the Burdizzo and rubber ring methods for castrating and tail docking lambs.

The behavioural and plasma cortisol changes in groups of six lambs, five to six days old, were used to compare the acute effects of four methods of castration and tail docking: Burdizzo, standard sized and small rubber rings, and a combined method in which the application of a standard rubber ring was followed immediately by the application of the Burdizzo just distal to it. A control group was also included. Active behaviours such as foot stamping and restlessness increased markedly only after the treatments with rubber rings. Less time was spent in abnormal postures, and the increases in plasma cortisol were least after the combined method. It was concluded that the lambs treated by all the methods suffered considerable acute pain, for up to three hours in some cases, but that the combined method produced the least pain as judged by the behavioural and physiological indices measured, and that the small rubber rings produced more intense pain for a shorter time than the standard rubber rings.

Castration of calves: a study of methods used by farmers in the United Kingdom.

A postal survey of farmers was conducted to determine the main methods used to castrate calves, and by whom and how they were applied. Among the 28 per cent of farmers who replied, those who did castrate calves used one or more of three methods: the Burdizzo was used by 43 per cent of farmers, surgery by 39 per cent, and rubber rings by 32 per cent, with 10 per cent using more than one method. Calves were castrated at all ages from less than one week to over six months, with one third of them being castrated at an age that legally requires the operation to be done under local anaesthesia by a veterinary surgeon. Rubber rings were never used by veterinary surgeons, but they carried out 43 per cent of surgical castrations, which was the method of choice in older calves. Local anaesthetic was used on 15 per cent of farms, mainly for surgical castrations. Sixty-seven per cent of farmers using the Burdizzo applied it twice, with the majority correctly applying the second crush below the first, and 90 per cent used precautions to control infection after surgical castration.

Effect of immunological castration on sexual and production characteristics in male cattle.

Ten male Friesian calves were actively immunised against luteinising hormone releasing hormone and their production compared with 10 Friesian steer calves. Five of the immunised calves responded poorly (group 1); the other five responded well (group 2), resulting in high antibody titres, low serum testosterone levels, involuted testes, reduced libido and semen production, and docile behavior. The castration effect lasted approximately six months before reversion to near normal male status. This temporary immunocastration failed to affect seriously performance until slaughter, and liveweight gain was substantially better in this group than in the steers. Carcase traits were similar in groups 1 and 2, but the lean content in both was considerably greater than in the steer carcases.

Response of aldosterone and blood pressure to angiotensin II infusion in anephric man. Effect of sodium deprivation.

Angiotensin II, infused intravenously, increased plasma aldosterone concentration in two of six anephric subjects taking their usual dietary quantities of sodium. After 3 days of dietary sodium restriction and weight-reducing hemodialysis, the aldosterone response to infused angiotensin II in the two previously reactive subjects was enhanced, while the four previously unreactive subjects also showed a rise in plasma aldosterone. Before and after sodium depletion the anephric subjects were less responsive than normal subjects. Even when sodium-depleted, the anephrics showed no further rise in plasma aldosterone when arterial plasma angiotensin II was increased by infusion to concentrations greater than 50-199pg/ml, in contrast to sodium-depleted normals who show progressive aldosterone responses with plasma angiotensin II concentrations up to at least 370pg/ml. Before the infusion of angiotensin II, arterial plasma renin, angiotensin II, and aldosterone were detectable in the anephrics, but were unchanged by dietary sodium restriction or weight-reducting hemodialysis. Sodium depletion caused significant falls in weight, plasma sodium, and blood pressure, but no changes in plasma potassium or cortisol. Increases in blood pressure in relation to increments of arterial plasma angiotensin II were unaffected by sodium depletion, as might be expected in the absence of a rise in endogenous angiotensin II.