Haematology, biochemistry and morphological features of peripheral blood cells in captive Boa constrictor.

The common boa (Boa constrictor) belongs to the family Boidae and represents one of the most popular traded and kept snake species in captivity. The early diagnosis, prevention and prophylaxis of diseases in this species, and in reptiles in general, still pose major challenges, also due to the lack of reliable reference values. This prompted us to conduct a study on clinically healthy captive B. constrictor to assess their basic health parameters in the blood (haematological and biochemical values, stress markers). Several parameters differed significantly between younger (<3 years) and older (≥3 years) boas; in the latter, the percentages of eosinophils, the haemoglobin and haematocrit levels, as well as the albumin and total protein levels, were higher. In male snakes, cholesterol levels were significantly higher than in females. Light and electron microscopy as well as immunohistochemistry served to identify and determine the morphological features of peripheral blood cells, that is, heterophils, basophils, eosinophils, azurophils, monocytes, lymphocytes, thrombocytes and erythrocytes. Leukocyte subpopulations, that is, T and B cells and monocytes, were also identified based on specific marker expression. The study provides data on haematological, biochemical and stress hormone levels, suitable as reference values, and on the blood cell morphology of B. constrictor which can serve as a guideline for further research on this species.

Estrus induction in white rhinoceros (Ceratotherium simum).

The estrous cycle length in the white rhinoceros (Ceratotherium simum) is either 4 or 10 wk. The cause(s) for this variation as well as the poor fertility rate in captivity remains under debate in this species. Most captive adult white rhinoceros undergo long anovulatory periods without luteal activity which are considered a major reason for their low reproductive rate. In this study, the synthetic progestin chlormadinone acetate (CMA) was tested in combination with hCG or the GnRH analogue deslorelin for its efficiency to induce ovulation in fourteen females without luteal activity and in three, regular cycling females. HCG (N = 12), injectable GnRH analogue (N = 8) and GnRH analogue implants (N = 15) were given to induce ovulation after CMA treatment. Treatment success was determined using both transrectal ultrasonography and progesterone metabolite EIA analysis. A preovulatory sized follicle (3.5 ± 0.1 cm) or a corpus luteum (5.1 ± 0.7) was present on the ovary one day after induction in 93.1% of the treatments. Despite this high rate of ovarian response, ovulation rate differed between the study groups. The ovulation rate for hCG, injectable GnRH analogue and GnRH analogue implants was 66.7%, 62.5% and 93.3%, respectively. Ovulation rate in cyclic females treated with GnRH implants was 100% (6/6) compared with 89% (8/9) in females without luteal activity receiving the same treatment. The length of the estrous cycle when induced with hCG was 4 wk (85.7%). The estrous cycle when induced with GnRH analogue was predominantly 10 wk long. Two females without luteal activity treated with GnRH became pregnant. In conclusion, CMA in combination with GnRH analogue implants was highly effective to induce ovulation in white rhinoceroses and thus can contribute to efforts aimed at increasing natural mating and reproductive rates in the captive white rhinoceros population.

High mitochondrial differentiation levels between wild and domestic Bactrian camels: a basis for rapid detection of maternal hybridization.

Hybridization between wild species and their domestic congeners often threatens the gene pool of the wild species. The last wild Bactrian camel (Camelus ferus) populations in Mongolia and China are examples of populations facing such a hybridization threat. To address this key issue in the conservation of wild camels, we analysed wild, hybrid and domestic Bactrian camels (Camelus bactrianus) originating from Mongolia, China and Austria. Through screening of an 804-base-pair mitochondrial fragment, we identified eight mitochondrial haplotypes and found high sequence divergence (1.9%) between C. ferus and C. bactrianus. On the basis of a mitochondrial DNA sequence fixed difference, we developed a diagnostic PCR restriction fragment length polymorphism (PCR-RFLP) assay to differentiate between wild and domestic camel samples. We applied the assay to 81 individuals and confirmed the origin of all samples including five hybrids with known maternal ancestry. The PCR-RFLP system was effective for both traditional (blood, skin) and non-invasive samples (faeces, hair), as well as for museum specimens. Our results demonstrate high levels of mitochondrial differentiation between wild and domestic Bactrian camels and that maternal hybridization can be detected by a rapid and reliable PCR-RFLP system.

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros.

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n=9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.

First successful artificial insemination with frozen-thawed semen in rhinoceros.

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of approximately 135 x 10(6) motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of approximately 500 x 10(6) motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals.

Reproductive endocrinology of the largest dasyurids: characterization of ovarian cycles by plasma and fecal steroid monitoring. Part I. The Tasmanian devil (Sarcophilus harrisii).

There is a strong body of knowledge on the reproductive endocrinology of macropods, but little detailed information is available on the hormonal control of reproduction in other marsupials. This study used plasma and fecal sex steroid monitoring to characterize the estrous cycle of the largest extant dasyurid-the Tasmanian devil (Sarcophilus harrisii). A pro-estrous pulse in plasma progesterone (1.33+/-0.2 ng/ml) occurred several weeks prior to onset of the luteal phase (LP), resulting in a characteristic biphasic pattern during the estrous cycle. This brief, pro-estrous progesterone pulse was associated with a predominantly cornified vaginal smear, and copulation in females paired with males. Mean luteal progesterone concentrations (5.28+/-0.8 ng/ml) were sustained and peaked around day 15 from luteal onset; thereafter, concentrations declined precipitously and returned to baseline around day 25. Females that did not produce young returned to estrus after 33.7+/-5.9 days. Fecal 20alpha-OH-pregnanes analyzed in a pregnanediol assay (PgD) were excreted in consistently higher levels than 20-oxo-pregnanes, but the pattern was similar for the two metabolites, and significantly correlated with fluctuations in plasma progesterone. Fecal total estrogen concentrations were highest during the follicular phase (FP) and accompanied a pro-estrous pulse in fecal progestagens. The mean duration of the estrous cycle was approximately 32 days, with a FP of around 14 days (range 8-23 days), and a luteal phase of around 18 days (range 12-25 days). There were no differences in the length of the LP between mated and non-mated cycles. Gestation length was 17.9+/-1.0 days (range 14-22 days). Fecal steroid monitoring revealed significant differences between the pattern of progestagens and estrogen concentrations during the pregnant and non-mated estrous cycle, suggesting maternal endocrine recognition of pregnancy in the Tasmanian devil.

Reproductive endocrinology of the largest Dasyurids: characterization of ovarian cycles by plasma and fecal steroid monitoring. Part II. The spotted-tailed quoll (Dasyurus maculatus).

Dasyurids exhibit a range of breeding patterns from semelparity through to an aseasonally polyestrous strategy, but detailed information on the reproductive endocrinology of many species is unavailable. This study aimed to extend our comparative understanding by characterizing the ovarian cycle of the spotted-tailed quoll (Dasyurus maculatus) through measurement of plasma progesterone, and also to investigate fecal sex steroid monitoring as an alternative, non-invasive technique. Longitudinal profiles revealed a biphasic pattern of plasma progesterone, with a significant pro-estrous pulse (0.97+/-0.3ng/ml) up to several weeks prior to onset of the luteal phase (LP). This pro-estrous period was associated with a predominantly cornified vaginal smear, onset of estrus behaviors and copulation. Mean luteal values for plasma progesterone were several fold higher (2.18+/-1.10 ng/ml) than during the follicular phase (FP) (0.75+/-0.02 ng/ml), and were sustained for approximately one month. Fecal progestagens and plasma progesterone were significantly positively associated during the estrous cycle. During the breeding period average concentrations of fecal total estrogens and pregnanediol (PgD) were significantly elevated. Ovarian activity during the FP was marked by increases in fecal estrogens, and rises in PgD which were sustained during the LP. In non-mated females the mean duration of the FP was significantly extended, being approximately twice as long (19.4+/-4.0 d) as for mated females (8.3+/-1.9 d) indicating coitus has some role in timing of ovulation in this species. This study has provided important new information on the reproductive biology of the female spotted-tailed quoll, and further demonstrated the usefulness of non-invasive endocrine techniques for monitoring ovarian cycles in marsupials.

Artificial insemination in the anoestrous and the postpartum white rhinoceros using GnRH analogue to induce ovulation.

The objective of this study was to develop AI and to achieve first time pregnancy in a nulliparous rhinoceros. For this, one 24-year-old irregular cycling female white rhinoceros was selected, which had never been mated. The endocrine function was monitored by faecal and serum pregnane analysis. Ultrasound determined the optimal day for AI by measuring follicle sizes of 2.0, 2.6, 3.0, 3.2 cm on days -6, -4, -1, 0 of the induced oestrous cycle, respectively. AI was performed and ovulation induced when a pre-ovulatory-sized follicle was present using GnRH analogue, deslorelin. Fresh semen was deposited in the uterine horn using a patented AI catheter overcoming the hymeneal membrane and torturous cervical folds non-surgically. Moreover, ultrasound monitoring of the uterine involution and ovarian activity on days 16, 26, 30 postpartum facilitated the induction of and the AI on the first postpartum oestrous in a rhinoceros using GnRH analogue. Two consecutive pregnancies were achieved by AI for the first time in the rhinoceros. Pregnancies were diagnosed by elevated serum and faecal 20-oxo-pregnane concentrations. In addition ultrasound measured biometric parameters of the two foetuses on days 86 and 133 of gestation. Two female calves were born after 490 and 502 days of gestation, yet one calf was stillborn. AI in rhinoceros might now be used as assisted reproduction technology tool to boost critically small captive rhinoceros populations.

High inter-individual variation in the gestation length of the hedgehog tenrec, Echinops telfairi (Afrotheria).

The gestation length (GL) of Tenrecs (Tenrecinae, Afrotheria) is still uncertain. This lack of knowledge also applies to the lesser hedgehog tenrec, Echinops telfairi, the species most commonly bred and maintained in captivity. The animals used in this study were held under controlled conditions (light, temperature and humidity). In order to determine the GL, groups of female tenrecs were subjected to various mating procedures followed by isolation periods of different lengths. A total of n=249 pregnancies were analysed and the number of offspring per litter was 3.29+/-0.09. The length of gestation could be determined in n=199 pregnancies and a mean GL of 67.53+/-0.36 days was calculated. Initial attempts with isolation periods of less than 16 days did not allow to accurately define the GL. Experiments with longer isolation periods and females subjected to only one mating procedure (n=10) revealed a variation in the GLs of 57-79 days. However, in one female a GL of only 50 days was also observed indicating an even greater range in GL variation. There was a statistically significant tendency for shorter GLs in the animals that conceived later in the mating season, but no statistical evidence was found that age, parity or litter size played an essential role in determining the GL. In conclusion, an unexpected high variability in gestation length in E. telfairi was demonstrated although the study animals were kept under controlled environmental conditions. The factors and mechanisms regulating this high intra-species variability in gestation length need further investigations.

Plasma, milk and faecal progesterone concentrations during the oestrous cycle of lactating dairy cows with different milk yields.

The hypotheses tested in this study were that neither average progesterone (P4) concentrations in plasma and milk nor average progesterone metabolites concentrations in faeces would differ during an oestrous cycle in two groups of cows with differing daily milk yields. High producing (HP = 8) and low producing (LP = 8) dairy cows were selected randomly for the study. Their oestrous cycles were initially synchronised using P4 and prostaglandin F2alpha. Chromic oxide capsules were administered twice daily to measure total faecal output. Samples of blood, faeces and milk were taken daily throughout one oestrous cycle, plasma and milk P4, and faecal progesterone metabolites (FP4M) assayed. The average daily milk yields in the two groups were 30.8 and 21.9l per day, respectively (P < 0.0001), although daily faecal output was similar in both the groups (HP, 7.7 versus LP, 6.9 kg DM; P = 0.24). Mean plasma and milk P4 concentrations were similar in both the groups (plasma P4, 4.12 versus 4.05 ng/ml; P = 0.3; milk P4, 8.2 versus 8.3; P = 0.9) during dioestrus. Average daily excretion of P4 to the milk was greater in HP than LP cows (252 versus 185 microg, P = 0.04). Neither concentration nor the daily yield of FP4Ms was affected by level of milk yield (concentration: 12.2 versus 11.5 microg/g; daily yield: 89.1 versus 82.9 mg per day; P > 0.05). These data showed that the concentrations of P4 in plasma and milk, and the concentrations and daily yields of FP4M were not affected by the level of daily milk yields which differed by about 41% of the LP average of 21.9l.

Effects of level of feeding and progesterone dose on plasma and faecal progesterone in ovariectomised cows.

The effects of two levels of feeding and two doses of progesterone (P4) on plasma and faecal progesterone metabolites (FP4M) were studied using a total of 24 ovariectomised (OVX), non-lactating, Holstein-Friesian cows. Cows were grazed on improved ryegrass/white clover pastures and allowed ad libitum access to pasture or were restricted to grazing for a total of 4 h per day in two 2 h periods. Progesterone (P4) was administered as one or two, simultaneous, intravaginal progesterone devices (CIDR). The cows were adapted to their pasture supply for 2 weeks before the start of the progesterone treatments. The progesterone devices were administered for 11 days and the cows were dosed with slow release chromic oxide capsules during the P4 treatment to allow faecal output (FO) to be estimated. Daily blood samples for P4 assay and weekly samples for blood metabolite assay were collected. Faecal samples were collected per rectum daily and assayed for pregnanes containing a 20-oxo-, 20alpha- or a 20beta-OH group by enzyme immunoassay (EIA). Daily FO was higher (P < 0.001) for ad libitum than pasture restricted cows (6.3 vs 4.1 kg DM) but was similar for both doses of P4. The average mass of P4 released from a CIDR device over a 11-day period was higher for cows allowed ad libitum pasture compared with those on restricted pasture (0.64 vs 0.60 g; P = 0.04). Plasma P4 concentrations, however, were higher in restricted than ad libitum fed cows (1x CIDR: 1.81 vs 1.41 ng/ml; 2x CIDR: 4.10 vs 3.46 ng/ml). Increasing the progesterone dose significantly (P < 0.001) increased both the concentrations and daily totals of the faecal pregnanes assayed and total FP4M. Restricted pasture cows had higher (P < 0.001) pregnanes and FP4M concentrations than cows fed ad libitum. Daily total faecal pregnane and FP4M did not differ between feeding levels except for faecal 20alpha-pregnane which was highest for ad libitum fed cows (P < 0.05). These results showed that the plasma concentrations of P4 in CIDR-treated OVX cows were negatively associated with the level of feeding. Level of feeding and dose of P4 affected the concentrations of FP4M, but the daily excretion rate of FP4M was not positively influenced by the level of feeding.

Progesterone clearance rate in lactating dairy cows with two levels of dry matter and metabolisable energy intakes.

The aim of these studies was to determine the effect of levels of dry matter (DM) and metabolisable energy (ME) intakes on clearance rate of progesterone (P4) in dairy cows. Thirty-two lactating Holstein-Friesian cows were selected for the study and were fed indoors in individual stalls for a period of 5 weeks. They were individually offered a diet of combinations of pasture, hay and pelleted cereal grain to achieve two different levels of DM and ME. In the first trial, 16 cows were allocated to two groups: (i) high DM (HDM), and (ii) low DM (LDM) intakes, while the amount of ME intake was constant. In the second trial, 16 cows were allocated to two groups: (i) high ME, and (ii) low ME intakes with similar amount of DM intake. A GnRH-agonist (deslorelin) was initially implanted in the ear of each cow to block endogenous P4 secretion. Then 3 weeks later, a CIDR device was inserted into the vagina of each cow and left in place for 11 days. Chromic oxide (Cr(2)O(3)) capsules were administered to allow daily faecal output (FO) to be estimated. Daily blood, faecal and milk samples were taken during the period of the experiment for P4 and faecal P4 metabolites analyses. Trial 1: The average milk yield was similar among cows in high and LDM intake groups (26.7 versus 25.0 l per day, P = 0.2). The average daily FO was 7.8 kg DM in the HDM and 5.7 in the LDM cows (P < 0.0001). Average daily DM intakes were 17.3 kg and 15.4 kg in the HDM and LDM groups, respectively (P < 0.0001). The average plasma P4 concentrations were similar between the two groups (1.56 versus 1.60 ng/ml, P = 0.7) but milk P4 concentrations were higher in LDM cows (4.6 versus 3.6 ng/ml, P = 0.02). The average daily excretion rate of P4 into the milk was higher in LDM cows (122.3 versus 88.5 microg, P = 0.002). The concentrations of faecal P4 metabolites (FP4M) were not influenced by the level of daily DM intake (2.85 versus 2.90 microg/g, P = 0.6). The average daily yields of FP4M were higher among cows in the HDM group (23.2 versus 16.3mg, P = 0.01). Trial 2: The average milk yield was 31.2l per day in HME cows compared to 25.0l per day in LME cows (P < 0.0001). The average daily FO was 7.8 kg DM in LME and 5.8 kg DM in HME cows (P < 0.0001), and the average DM content of faeces was higher in LME cows (15.8 versus 12.7%, P = 0.01). The average daily ME intake was 213MJ per day in HME group compared to 183MJ per day in LME group (P<0.0001). The average plasma and milk P4 concentrations were similar between the two groups (plasma P4 = 1.54 versus 1.56 ng/ml, P = 0.4; milk P4: 3.7 versus 3.6 ng/ml, P = 0.6). The average daily excretion rate of P4 into the milk was higher in HME cows (114 versus 88.5 microg, P = 0.03). Concentrations of FP4M were not influenced by the level of daily ME intake (2.5 versus 2.85 micro g/g, P = 0.08). However, daily yields of FP4M were greater in the LME group (23.2 versus 14.4 mg, P = 0.01). In conclusion, this study was unable to establish a relationship between the level of DM and ME in the diet with the excretion rates of FP4M metabolites and plasma P4 concentrations.

Faecal progesterone metabolites and behavioural observations for the non-invasive assessment of oestrous cycles in the common wombat (Vombatus ursinus) and the southern hairy-nosed wombat (Lasiorhinus latifrons).

Wombats belong to Australia's unique marsupial species. Two of the three remaining species, the common wombat (Vombatus ursinus) and the southern hairy-nosed wombat (Lasiorhinus latifrons) are abundant. The third species, the northern hairy-nosed wombat (Lasiorhinus krefftii) has only about 115 individuals left in the wild. This study aimed to gain further insight into the basic reproductive biology of wombat species and evaluate the value of faecal progesterone metabolites and behavioural patterns as a means for non-invasive monitoring of the oestrous cycle in common and the southern hairy-nosed wombats. In an initial study, three different faecal steroid assays showed that 20alpha-OH-pregnanes were the main progesterone metabolites. These metabolites were examined in captive female common wombats (n = 5) and southern hairy-nosed wombats (n = 2). In one female common wombat 11.7 days with a follicular phase of 25.6 +/- 6.3 days and a luteal phase of 28.2 +/- 12.7 days. The data for faecal pregnanes obtained in the southern and in one male common wombat oestrous related behavioural data were obtained. Individual cycling females exhibited a significant relationship between plasma progesterone and faecal pregnanes. In the common wombat, the values for faecal pregnanes showed an oestrous cycle length of 55.1 +/- hairy-nosed wombat during the breeding season gave an oestrous cycle length of 41.1 +/- 12.8 days with a follicular phase of 27.9 +/- 12.3 days and a short luteal phase of 13.3 +/- 1.1 days. The behavioural data show that the faecal sniffing behaviour of the male, tended to increase around the time that oestrous was found. In conclusion, monitoring of 20alpha-OH-pregnanes in wombat faeces could be a useful methodology to monitor reproductive cycles in the wombat, and can possibly be applied to monitor the endangered northern hairy-nosed wombat.

Suppression of ovarian progesterone production in dairy cows using an implant of GnRH-agonist (deslorelin) for the purpose of evaluating progesterone metabolism [corrected].

OBJECTIVE: To evaluate the potential of an implant of a GnRH-agonist (deslorelin) to create a progesterone free animal suitable for studying progesterone (P4) metabolism in intact cows by measuring blood P4 and faecal P4 metabolites. METHODS: Experiment 1: Eighteen non-lactating cycling Holstein-Friesian cows, 4 to 7 years old, were allocated to one of three groups to study plasma P4 concentrations preceding an intravaginal insert. These groups comprised: i) a deslorelin group (GnRH-agonist implanted); ii) a PGF group receiving two injections of prostaglandin (PGF2alpha) 12 days apart; and, iii) an ovariectomised (OVX) group. An intravaginal device (CIDR) was inserted into the vagina of each animal and left in place for 11 days. Plasma P4 concentrations were measured during the study period. Experiment 2: Twelve non-lactating cycling Holstein-Friesian cows, 4 to 7 years old, were allocated to two groups: i) a deslorelin group (GnRH-agonist implanted); and ii) an ovariectomised group. Plasma P4 and faecal P4 metabolites (20-oxo-pregnanes, 20alpha-OH and 20beta-OH) were monitored for a period of 5 weeks. RESULTS: Experiment 1: Average plasma P4 concentration did not differ between the three groups (1.28, 1.43 and 1.55 ng/mL for deslorelin, OVX and PGF cows, respectively, P = 0.8) during the period of supplementation. Experiment 2: There was no difference in plasma P4 (mean plasma P4 < 0.02 ng/mL, P = 0.9) and faecal P4 metabolites between deslorelin and OVX cows 2 weeks after the implantation (P = 0.7). CONCLUSIONS: These data showed that a GnRH-agonist (deslorelin) implant may be used as an alternative to ovariectomy to create a progesterone free animal suitable for studying the metabolism of administered P4.

The effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows.

The objective of the present study was to determine the effect of level of feed intake of pasture on P4 clearance rates in dairy cows. Twelve non-lactating Holstein-Friesian cows aged 4-9 years were randomly allocated to a restricted or ad libitum group. The ad libitum group had unrestricted access to irrigated pasture, whereas the restricted group had access for only 2h per day. Each animal was drenched orally twice daily with a chromic oxide capsule to allow daily feed intake to be estimated from faecal output (FO). Endogenous progesterone (P4) production was eliminated by subcutanously implanting a capsule containing 6 mg of a potent GnRH-agonist (deslorelin) into the ear of each animal 3 weeks before inserting a CIDR device containing 1.9 g P4 into the vagina. Two luteolytic PGF2alpha were given 10 days later. Each device was removed after 11 days and residual P4 measured. Daily plasma samples were assayed for P4. Faecal samples were also taken daily and assayed for pregnanes (FP4M) containing a 20-oxo-, a 20alpha- or a 20beta-OH group with EIAs. The average daily dry matter (DM) intake of pasture was higher for cows in the ad libitum group (15.9 versus 6.3 kg DM, P=0.001). Their plasma P4 concentrations were lower (1.08 versus 1.71 ng/ml, P=0.05), even though the average residual P4 content of the used CIDR devices was not affected by feed intake (1.20 versus 1.25 g, P>0.05). The concentrations of FP4M were not affected by level of feed intake (20-oxo-: 3.3 versus 1.7, 20alpha-: 3.5 versus 3.7, 20beta-: 2.1 versus 3.2 microg/g DM). Daily excretion rates of 20-oxo- and 20alpha- were higher in ad libitum cows (20-oxo-: 17.8 versus 4.3mg per day, P=0.05; 20alpha-: 18.2 versus 8.9 mg per day, P=0.001), but daily yield of faecal 20beta- was not affected by feed intake (11.9 versus 8.6 mg per day, P=0.5). These results show that there was a negative relationship between feed intake and plasma P4 concentrations in these CIDR-treated GnRH-downregulated Holstein cows. Concentrations of FP4M were not affected by level of feed intake or FO, but daily excretion rate of FP4M was associated with the volume of faeces.

Progesterone metabolism in ovariectomised non-lactating Holstein-Friesian cows treated with progesterone with two levels of feed intake.

The goal of this study was to measure the effects of level of feeding and the form of progesterone (P4) administration on the concentrations and yields of faecal P4 metabolites relative to differences in plasma P4 concentrations in non-lactating cows. Six non-lactating Holstein-Friesian cows were ovariectomised (OVX) and allocated to two groups: (i) P4 by subcutaneous injection (P4-s.c., n=3); and (ii) P4 administration per vaginum (P4-p.v., n=3). Each cow in the P4-s.c. group was injected subcutaneously once daily with 200mg P4. Each cow in the P4-p.v. group had a CIDR device inserted for 11 days when it was removed and replaced with a second device for further 11 days. Cows were fed a ration containing lucerne (33%) and oaten (66%) chaff at a maintenance level (M) in two portions in the first period of the study, and at a half-maintenance (1/2M) level during the second period. Chromic oxide capsules (Cr(2)O(3)) were administered twice daily to allow faecal output (FO) to be estimated. Plasma P4 and faecal P4 metabolites (FP4M; 20-oxo-pregnanes, 20alpha- and 20beta-OH-pregnanes) were measured during the treatment period. Daily FO declined after reducing the M diet to 1/2M (4.77 versus 2.61kg; P<0.01), whereas plasma P4 concentrations increased in the P4-s.c. group (4.2 versus 6.2ng/ml; P<0.05), but not in the P4-p.v. group (0.9 versus 1.0ng/ml; P>0.2). The mass of P4 released from a CIDR device during each 11-day period (M or 1/2M) was similar (0.66 versus 0.63g). Faecal 20-oxo-pregnanes (20-oxo-) concentrations were not affected by day or level of feeding, whereas faecal 20alpha-OH (20alpha-) and 20beta-OH (20beta-) concentrations were increased with the 1/2M diet in the P4-s.c. group (4.3 versus 5.6 microg/gDM; 2.2 versus 5.6 microg/gDM, respectively; P<0.05), but not in the P4-p.v. group (2.3 versus 2.7 microg/gDM; 1.7 versus 3.04 microg/gDM P>0.05). These changes in concentration only partly compensated for the reduced FO with the 1/2M diets as daily yields of FP4M (20-oxo- and 20alpha-) were greater during the M diet period (20-oxo-: 6.9 versus 4.1 microg/gDM; 2.7 versus 1.5 microg/gDM, for P4-s.c. and P4-p.v. groups, respectively; P<0.05, 20alpha-: 19.9 versus 13.6 microg/gDM; 10.9 versus 6.6 microg/gDM for P4-s.c. and P4-p.v. groups, respectively; P<0.05). The level of feeding and the route of P4 administration had key roles in controlling P4 concentrations in blood and daily FP4M yield.

Excretion rate of progesterone in milk and faeces in lactating dairy cows with two levels of milk yield.

This study was conducted to measure the effect of the level of daily milk yield on the excretion rate of progesterone (P4) in milk and faeces in high-producing (HP) and low-producing (LP) lactating dairy cows. A GnRH-agonist was implanted to block endogenous production of P4. A CIDR device was inserted into the vagina and left in place for 11 days. The average and peak milk yields were greater in HP cows (P < 0.0001). Mean plasma concentrations of P4 were also similar in both groups (P = 0.44), even though the average mass of P4 delivered from a CIDR device was higher with HP cows (P = 0.02). Average milk P4 concentration was similar in both groups (P = 0.81), so that average daily excretion of P4 in the milk was greater with HP cows (P = 0.05). The concentrations (P = 0.83) and daily yields (P = 0.4) of total faecal progesterone metabolites were not affected by level of milk yield. These data show that the concentrations of plasma and milk P4, and the concentration and yield of P4 metabolites are not affected by the levels of daily milk yield.

Fecal progesterone, estrogen, and androgen metabolites for noninvasive monitoring of reproductive function in the female Indian rhinoceros, Rhinoceros unicornis.

This investigation aimed to establish noninvasive methods for endocrine monitoring of estrous cycles and pregnancy in the Indian rhinoceros. Fecal samples were collected 1-3 times per week from nonpregnant and pregnant captive females (n = 7). Enzyme immunoassays for fecal progesterone, androgen, and estrogen metabolites, respectively, were tested for their ability to determine follicular and luteal phases and to characterize endocrine profiles during pregnancy. Antibodies used were raised against pregnanediol (20 alpha-OH-pregnanes), 20-oxo-pregnanes, epiandrosterone (17-oxo-androstanes), and total estrogens. Androgens and estrogens were found to be reliable indicators of the follicular phase, whereas 20 alpha-OH- and 20-oxo-pregnanes were reliable indicators of luteal function. Progesterone metabolites were also reliable indicators of pregnancy, whereas 17-oxo-androstanes and estrogens were basal throughout gestation. Estrous cycles were regular throughout the year, with an average cycle length of 43.4 +/- 1.5 (n = 27) days; the length of the follicular phase, as indicated by elevated estrogen levels, was 15.9 +/- 1.0 days, whereas the luteal phase, as indicated by elevated 20-oxo-pregnane levels, was 19.1 +/- 0.4 days. Fecal pregnane values were already increasing while follicular estrogen values were still decreasing. The length of the diestrus, indicated by basal steroid levels between declining 20-oxo-pregnanes and subsequently increasing estrogens, was 11.4 +/- 1.2 days. Pregnane levels increased from the 3rd month of gestation onward and levels exceeded luteal phase concentrations approximately 10 times by the 7th month of gestation onward. HPLC separation of immunoreactive fecal metabolites indicated the presence of estrone, estradiol-17beta, and several 17-oxo-androstanes, 20 alpha-OH-pregnanes, and 20-oxo-pregnanes. Concentrations of a peak with an elution profile similar to that of pregnanediol increased as pregnancy progressed. Postpartum fecal estrogen and 17-oxo-androstane concentrations in one animal indicated follicular development comparable to the follicular phase of the estrous cycle, but this was not followed by a subsequent luteal phase. In conclusion, estrous cycle and pregnancy in Indian rhinoceroses can be monitored using fecal steroid analysis. Pregnane metabolites were reliable indicators of the corpus luteum and pregnancy, whereas fecal 17-oxo-androstanes and estrogens were indicators of the follicular phase.

Noninvasive reproductive monitoring in the okapi (Okapia johnstoni).

Fecal progestagen analysis in okapis (Okapia johnstoni) was used for diagnosis of pregnancy and reproductive disorders, including a comparison of urinary and fecal progestagen analysis and endocrine data on the postpartum period. Data were generated on reliability of fecal progestagen analysis in early pregnancy diagnosis, and case reports were compiled involving single animals with missing luteal activity, abortion after twin pregnancy, and abortions due to deficient placental progestagen production. There was approximately 100-200-fold higher progestagen concentration in feces than in urine, thus explaining the high reliability of fecal progestagen evaluations in diagnosing luteal function and pregnancy. The postpartum period was characterized by lactational anestrus of several months duration, and a postpartum estrous cycle about 2-3 wk after parturition was observed in two of eight animals. An animal with five abortions due to deficient placental progestagen production was treated with altrenogest in a subsequent pregnancy and carried the fetus to term.