Franz Schwarzenberger

Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals

Abstract Non-invasive faecal oestrogen and progesterone metabolite evaluations are well established approaches for monitoring reproductive function in a variety of mammalian species. The route of excretion of steroid hormone metabolites varies considerably among species, and also between steroids within the same species. Steroid concentrations in faeces exhibit a similar pattern to those in plasma, but have a lag time, which depending upon the species, can be from 12 h to more than 2 days. Faecal steroid metabolites in mammals are mainly unconjugated compounds. Faecal oestrogens consist predominantly of oestrone and/or oestradiol-17α or -17β. Therefore, specific oestrogen antibodies or antibodies against total oestrogens can be used for their determination. Progesterone is metabolised to several 5α- or 5β-reduced pregnanediones and hydroxylated pregnanes prior to its faecal excretion. Therefore, relevant antibodies for their determination show considerable cross-reactivities with several pregnane metabolites, whereas specific progesterone antibodies are less suitable. Faecal oestrogen evaluations have been used as reliable indicators of pregnancy in several ungulate and some primate species. They have also been used to determine the preovulatory period in carnivores, corpus luteum activity in New World primates, and to diagnose cryptorchidism in horses. Faecal progesterone metabolite analysis has been successfully used for monitoring corpus luteum function and pregnancy, abortion, seasonality and treatment therapies in an ever expanding list of species.

Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group‐specific enzyme immunoassays for 20‐oxo‐pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody.

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.

Faecal progesterone metabolite analysis for non-invasive monitoring of reproductive function in the white rhinoceros (Ceratotherium simum).

The two subspecies of white rhinoceros, southern (Ceratotherium simum simum) and northern (Ceratotherium simum cottoni), breed poorly in captivity, and estimates of oestrous cycle length vary considerably (range, 25-90 days). To characterise reproductive patterns, faecal samples were collected 2-3 times/week for up to 56 months from non-pregnant animals (n=21) of both subspecies. Immununoreactive pregnanes containing a 20-oxo-group (20-oxo-P) were analysed in a group-specific enzyme immunoassay using an antibody against 5alpha-pregnane-3beta-ol-20-one 3HS:BSA. Reproductive patterns were highly variable among and within individual animals. However, rhinoceroses could be classified into four major categories on the basis of oestrous cycle length and luteal phase 20-oxo-P concentrations: (1) regular oestrous cycles of 10 weeks duration and > 800 ng/g (n=2 animals); (2) oestrous cycles between 4-10 weeks and 250-750 ng/g (n=6); (3) no apparent cycle regularity, but luteal activity indicated by 20-oxo-P concentrations of 100-200 ng/g (n=6); (4) no apparent luteal activity as indicated by 20-oxo-P of < 100 ng/g (n=7). In two attempts to induce ovarian activity, chlormadinone acetate was fed daily to one animal for 35 and 45 days, respectively. Each treatment was followed by a subsequent hCG injection which resulted in luteal phases of 17 and 18 days, respectively, beginning about 10 days after hCG. Concentration of faecal 20-oxo-P in one pregnant animal during the 4th and 5th month of gestation were markedly higher than those observed during the luteal phase of the cycle. In conclusion, two thirds of white rhinoceroses in this study had erratic or missing luteal activity, whereas variable cycles of 4-10 weeks in length were evident in six females, and regular oestrous cycles of 10 weeks in length were found in two animals.

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.

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 Australias 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.

USE OF GROUP-SPECIFIC ANTIBODIES TO DETECT FECAL PROGESTERONE METABOLITES DURING THE ESTROUS CYCLE OF COWS

Abstract Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P

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.

Analysis of the mitochondrial genome of cheetahs (Acinonyx jubatus) with neurodegenerative disease

Abstract The complete mitochondrial genome of Acinonyx jubatus was sequenced and mitochondrial DNA (mtDNA) regions were screened for polymorphisms as candidates for the cause of a neurodegenerative demyelinating disease affecting captive cheetahs. The mtDNA reference sequences were established on the basis of the complete sequences of two diseased and two nondiseased animals as well as partial sequences of 26 further individuals. The A. jubatus mitochondrial genome is 17,047-bp long and shows a high sequence similarity (91%) to the domestic cat. Based on single nucleotide polymorphisms (SNPs) in the control region (CR) and pedigree information, the 18 myelopathic and 12 non-myelopathic cheetahs included in this study were classified into haplotypes I, II and III. In view of the phenotypic comparability of the neurodegenerative disease observed in cheetahs and human mtDNA-associated diseases, specific coding regions including the tRNAs leucine UUR, lysine, serine UCN, and partial complex I and V sequences were screened. We identified a heteroplasmic and a homoplasmic SNP at codon 507 in the subunit 5 (MTND5) of complex I. The heteroplasmic haplotype I-specific valine to methionine substitution represents a nonconservative amino acid change and was found in 11 myelopathic and eight non-myelopathic cheetahs with levels ranging from 29% to 79%. The homoplasmic conservative amino acid substitution valine to alanine was identified in two myelopathic animals of haplotype II. In addition, a synonymous SNP in the codon 76 of the MTND4L gene was found in the single haplotype III animal. The amino acid exchanges in the MTND5 gene were not associated with the occurrence of neurodegenerative disease in captive cheetahs.

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.