Progesterone, pregnanediol-3-glucuronide, relaxin and oestrone sulphate concentrations in saliva, milk and urine of female alpacas (Vicugna pacos) and their application in pregnancy diagnosis

The pregnancy-associated hormones, progesterone (P4), pregnanediol-3-glucuronide (PdG), relaxin (RLN) and oestrone sulphate (E1S) in plasma, saliva, milk and urine of alpacas were measured in order to assess their potential use for pregnancy diagnosis. Samples were obtained from 36 female alpacas before mating and at different stages throughout pregnancy (confirmed by ultrasonography). The hormone concentrations were determined using enzyme immunoassays. Milk samples were also tested using a commercial on-farm P4 kit, designed for dairy cattle. Although the concentration of P4 in plasma, milk and urine, and the concentration of PdG in urine were significantly higher in pregnant than in non-pregnant alpacas, there was no difference in the concentrations of P4 or PdG in saliva. The on-farm milk P4 kit showed a sensitivity of 90 per cent for diagnosis of pregnancy and a specificity of 69 per cent for non-pregnancy. The concentration of RLN in plasma increased significantly after the second month, and concentration of E1S in plasma and urine during the last month of pregnancy, whereas, there were no significant differences in RLN or E1S concentrations in saliva and milk between pregnant and non-pregnant alpacas. Values of P4, RLN and E1S in plasma, and PdG and E1S in urine are comparable with the previous reports in alpacas and, therefore, can be confirmed as an indicator for pregnancy. This is the first study to include determination of pregnancy-associated hormones in the saliva and milk of alpacas. However, saliva seems to be unsuitable for pregnancy diagnosis in alpacas, whereas, P4 in milk, as well as PdG and E1S in urine, seem to be adequate tools for diagnosis.     

Refinement of a commercial bench-top relaxin assay for pregnancy diagnosis using urine from domestic and nondomestic felids

Relaxin, a 6-kDa polypeptide hormone, is excreted in the urine during pregnancy in several mammalian species. A recent study showed that detection of urinary relaxin using a bench-top serum assay (Witness relaxin kit, Synbiotics Corp., San Diego, California 92127, USA) can be diagnostic for pregnancy in domestic cats (Felis silvestris catus), but it is unknown whether the bench-top kit is applicable with urine across felid species. Our objectives were to 1) examine modifications in urine processing to improve kit reliability in pregnant cats, 2) evaluate the impact of concentrating urine via filtration on relaxin detection, 3) assess the effect of sample freezing on relaxin concentrations, and 4) begin quantifying urinary relaxin levels in nondomestic felids. Urine and serum were collected from domestic cats and nondomestic cat species (Pallas' cat, Otocolobus manul; sand cat, Felis margarita; cheetah, Acinonyx jubatus; and lion, Panthera leo) at several times after breeding. Urine samples, subjected to various processing methods, were tested using the bench-top kit, and relaxin levels were later quantified via radioimmunoassay. For domestic cat urine samples, filtration and addition of protein/phosphate buffer improved the consistency of the relaxin kit for early pregnancy diagnosis. Urine freezing caused a slight (approximately 13%) but significant decrease in relaxin concentrations, but frozen-thawed samples still tested positive with the bench-top kit. In nondomestic felids, urinary relaxin immunoreactivity during pregnancy was similar to or higher than that of pregnant domestic cats, suggesting that relaxin is a reliable cross-species marker of pregnancy. Urinary relaxin was detectable using the bench-top kit in pregnant Pallas' cats, but urine samples from other species tested negative, regardless of processing methods. Findings suggest that measurement of urinary relaxin is a promising approach for noninvasive pregnancy diagnosis in exotic felids, but further assessment of urinary relaxin profiles among cat species and modification of the bench-top relaxin kit are warranted to improve cross-species utility.     

Comparison of plasma progesterone, transrectal ultrasound and pregnancy specific proteins (PSPB) used for pregnancy diagnosis in reindeer

The study aimed to compare plasma progesterone concentrations, rectal ultrasonography and plasma concentrations of pregnancy-specific protein B (PSPB) used for pregnancy diagnosis in reindeer. A total of 1,595 blood plasma samples were collected between 1991 and 1996 from 3 semidomestic reindeer (Rangifer tarandus tarandus) herds on the Norwegian mainland (Mager?y, S?r?y, Filefjell) and from 92 wild Svalbard reindeer (Rangifer tarandus platyrhynchus). Samples were collected between January and late April. Plasma levels of progesterone and PSPB were measured and used as indicators of pregnancy. In addition, animals from the Filefjell herd and the Svalbard reindeer were investigated using transrectal ultrasound. The results showed that plasma progesterone lower than 7 nmol l-1 rarely occurs in females diagnosed pregnant either by ultrasound or by observing a calf at foot 7 months after blood sampling. A very good agreement was found between plasma progesterone and PSPB when used for pregnancy diagnosis. On the Norwegian mainland, but not to the same extent on Svalbard, a high proportion of females with a high progesterone concentration was diagnosed not pregnant by ultrasound. This probably reflects a high rate of false negative diagnoses by the ultrasound method rather than false positives in the progesterone analysis.     

奶牛发情周期中毛 唾液和乳汁孕酮水平的变化

本实验采用放射免疫分析法(RIA)测定了11头奶牛一个发情周期里毛、唾液、乳汁中孕酮(P_4)含量。经产牛(n=8)的毛和唾液中P_4水平变化与乳汁中P_4水平变化一致。青年牛(n=3)的毛和睡液中的P_4水平变化一致,并分别与经产牛毛和唾液中P_4水平变化一致。7头配种后的经产牛和5头配种后的青年牛分别取20和23天的样品进行妊娠诊断,与配种后60天的直检结果相对照,依据经产牛毛、乳P_4水平进行妊娠诊断的阳性准确率分别是85.7％(6/7)和100％(7/7),依据青年牛毛P_4水平进行妊娠诊断的阳性准确率为100％(5/5)。     

Vaginal electrical resistance in cows: 2. Relationship to milk progesterone concentrations during the reproductive cycle

When the vaginal electrical resistance (VER) was measured in the anterior vagina in 4 cows during a total of 12 oestrous cycles, there was a close correlation between VER, milk progesterone levels and visual observations of oestrus. The cyclic changes in both VER and milk progesterone ceased in 2 cows which became pregnant during the study. In a pregnant cow, the VER was found to be constant both pre-partum and immediately post-partum. These results show that measurement of VER can be a useful aid in the confirmation of oestrus and suggest that the technique may also be applicable to the early diagnosis of pregnancy.     

Pregnancy Diagnosis in Cats Using a Rapid, Bench-top Kit to Detect Relaxin in Urine

Relaxin is a pregnancy-specific hormone in the queen and is produced by the placenta. Both serum and urinary relaxin levels can be used to diagnose and monitor pregnancy in the cat; however, only serum levels are commonly measured in practice. The present study aimed to assess whether urine could be used for the rapid diagnosis of pregnancy at an early stage in domestic cats using a bench-top kit to detect relaxin. Paired serum and urine samples were collected during the first month of gestation in six cats. The samples were tested by applying neat serum, urine or urine diluted in non-pregnant cat serum to the Witness Relaxin kit. Relaxin concentrations in the paired samples were also measured by radioimmunoassay. All undiluted urine samples from pregnant cats tested negative using the bench-top kit; however, the kit was able to detect relaxin in urine after dilution with non-pregnant cat serum. Using this as the test sample, the kit was accurate at diagnosing pregnancy from 28 days after mating and some samples tested positive at 21 days after mating. This preliminary work could lead to the development of a home pregnancy test for cats.     

Progesterone concentration in defatted milk of dairy cows in early pregnancy.

Progesterone concentrations have been measured in defatted milk of British Friesian cows of four herds during the oestrus cycles (other than short cycles) immediately before artificial insemination (AI) at oestrus and immediately after AI (in non-pregnant cows), and during early pregnancy. Differences in mean progesterone concentrations between herds were significant (P less than 0.05) on all days within the day 10-18 period after AI, both in pregnant and in non-pregnant, inseminated cows but were not significant between pregnant and non-pregnant cows within herds until day 17 or 18. It is concluded that up to this time (that of luteolysis in non-pregnant cows) undefined factors, variable among herds, can have a much greater influence on the rate of progesterone secretion by corpora lutea and consequent progesterone concentration in plasma and milk than does the presence of conceptuses. Maximum mean progesterone concentration reached during early pregnancy in two herds did not differ significantly; it was reached in the 11-15-day period in one herd but not until 46-50 days in the second. Mean progesterone concentration declined after day 90.     

Serum relaxin and progesterone concentrations in pregnant, pseudopregnant, and ovariectomized, progestin-treated pregnant bitches: detection of relaxin as a marker of pregnancy

Serum concentrations of relaxin and progesterone were measured by specific radioimmunoassays in pregnant, pseudopregnant, or ovariectomized (between gestation weeks 4 and 5) pregnant Labrador Retriever bitches. Daily administration of 17 alpha-ethyl-19-nortestosterone was performed to maintain gestation in the ovariectomized pregnant bitches. This synthetic gestagen was selected because it did not interfere with the assay for endogenously secreted progesterone concentration in serum. Serum progesterone concentration was high in ovarian-intact pregnant or pseudopregnant bitches, but the mean progesterone concentration in pseudopregnant bitches (evaluated at 4 weeks after mating) was only 56% of the concentration in pregnant bitches. After ovariectomy, serum progesterone concentration decreased to undetectable values. Unlike progesterone, serum relaxin concentration increased during the latter half of pregnancy in the ovarian-intact and in the ovariectomized pregnant bitches, but relaxin was not detectable at any time in the pseudopregnant bitches. The amount of relaxin measured in the ovariectomized pregnant bitches was less (P less than 0.05) than that in ovarian-intact bitches, suggesting that the ovaries may have contributed to the total circulating relaxin concentration in the latter. Placental production of relaxin might have accounted for the serum relaxin concentration after ovariectomy; thus, the ovary and placenta each may secrete relaxin during gestation in bitches. Regardless of its source, measurement of serum relaxin concentration may offer a useful way of distinction between pregnancy and pseudopregnancy in dogs.     

Milk progesterone determination in buffaloes post-insemination

Milk progesterone levels were studied in 17 buffaloes between post-insemination and 30 days thereafter. Six animals were confirmed pregnant. Seven of 10 animals took greater than 24 days to return to oestrus and the pattern of change of progesterone levels indicated a steady increase from 1 ng/ml on day 2 to 20 ng/ml on day 14 post-insemination, falling subsequently to about 5 ng/ml, 2 days prior to oestrus. In pregnant buffaloes, the milk progesterone levels showed a continuous increase up to 20 days post-insemination but did not decline thereafter. In individual buffaloes returning to oestrus, a cut-off milk progesterone level of greater than 10 ng/ml was considered for pregnancy diagnosis, 20, 22 and 24 days post-insemination. The test was 60, 75 and 75% accurate for detection of pregnancy on days 20, 22 and 24 respectively and 100% for non-pregnancy diagnosis on all three days. Individual animals showed a highly variable oestrous cycle length, which has been suggested as a contributory factor for false pregnancy diagnosis by milk progesterone test in this species. Milk progesterone assay may also identify silent heats, and clinical disorders.     

Comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk

The objective of this study was to compare 2 enzyme immunoassays (EIAs) with a radioimmunoassay (RIA) as to sensitivity and accuracy in the measurement of the progesterone (P4) concentration in bovine plasma, skim milk, and whole milk. The 72 samples from 24 lactating dairy cows expected to have either a high P4 concentration (cows in diestrus or pregnant) or a low P4 concentration (cows in estrus or anestrus) were analyzed by RIA, solid-phase EIA (SPEIA), which included a solvent extraction step, or direct EIA (DEIA) without solvent extraction. The overall mean concentrations of P4 did not differ (P < 0.4) among the assays. However, for the cows that were in diestrus or pregnant, the mean P4 concentrations (and standard error) were higher (P < 0.03), regardless of sample type, with RIA than with SPEIA, at 7.3 (0.7) and 6.1 (0.6) ng/mL, respectively. When only the high-P4 samples analyzed by RIA were compared, the mean P4 concentration was higher (P < 0.001) in whole milk than in skim milk, at 9.8 (1.0) and 4.1 (0.7) ng/mL, respectively. Although the mean P4 concentrations in the low-P4 samples did not differ (P < 0.80) among assays, the proportions of cows with a P4 concentration > or = 1 ng/mL were 3%, 14%, and 44% for RIA, SPEIA, and DEIA, respectively (P < 0.01; DEIA > SPEIA > RIA).     

奶牛早孕诊断乳汁孕酮临界值的确定

随机选择健康荷斯坦母牛６３头，其中发情周期牛５头，配后１８－２４天牛３０头和通过直接确定的怀孕牛２８头，按程序采集末乳乳样。用放射免疫测定法（ＲＬＡ）测定乳汁孕酮（ＭＰ４）浓度。以发情第３天ＭＰ４浓度加上２倍标准差为未孕临界值的土限，以配后１８－２４天怀孕牛ＭＰ４浓度的平均值减去１倍标准差为怀孕临界值的下限。结果表明，奶牛ＭＰ４早孕诊断临界值为：≥７．１９ｎｇ／ｍｌ为怀孕，〈４．７２ｎｇ／ｍｌ为未     

Use of an ELISA for plasma progesterone to facilitate rabbit husbandry

There is a need for a simple and accurate method for detecting pregnancy in rabbits; the available methods are not ideal and may not provide the diagnosis at an appropriate time for the early remating of non-pregnant animals. This paper describes the use of an ELISA kit to measure progesterone concentrations in rabbit plasma; a qualitative assessment of the results appears to be sufficiently accurate for pregnancy diagnosis, as does the use of serum instead of plasma. The technique can be used to predict ovulation and to distinguish between pregnant and pseudopregnant animals.     

Reliability of pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulfate

The pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulphate in the blood serum without sample extraction is described. It was found that for the pregnancy diagnosis the period between days 22 and 30 of pregnancy can be used since in this period the estrone-sulphate concentrations in all pregnant sows markedly exceeded those of 64 non-pregnant animals. The estrone-sulphate estimation cannot be used for the pregnancy diagnosis before day 22 and between day 30 and 40 because the estrone-sulphate concentrations in most samples collected in these periods were lower than 4 nmol.l-1.     

Measurement of progesterone in sheep using a commercial ELISA kit for human plasma

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze–thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.     

Pregnancy diagnosis by milk progesterone on days 18, 22, and 24 postbreeding in dairy cows

Milk samples were collected from 91 dairy cows on days 18, 22, and 24 postbreeding. The progesterone values of these samples were assayed, using enzyme-immuno-assay for pregnancy diagnosis. These animals were classified by nonpregnant (low progesterone values, 5 ng/ml or less) and pregnant (high progesterone values, more than 5 ng/ml). The accuracy of diagnosis on days 18, 22, and 24 postbreeding were 56%, 78%, and 79% for pregnant cows and 90%, 93%, and 93% for nonpregnant animals. The sampling date had a significant effect on diagnostic accuracy (P less than 0.05).     

The effects of gonadotrophin releasing hormone administration four days after insemination on first-service conception rates and corpus luteum function in dairy cows.

One hundred and eighty-five Holstein-Friesian dairy cows received either sterile water or 250 micrograms of gonadotrophin releasing hormone intramuscularly on the fourth day after the first service postpartum. Heparinized blood samples were taken immediately prior to treatment (day 4) and on day 8 postinsemination for analysis of plasma progesterone concentration. Pregnancy diagnosis was carried out by rectal palpation at 42 days postinsemination and reconfirmed after 60 days postbreeding. The pregnancy rates after first, second or third service were not significantly different between gonadotrophin releasing hormone-treated and control cows. Plasma progesterone concentrations on day 4 and day 8 postinsemination, as well as the change in plasma progesterone concentration from day 4 to day 8, were similar for gonadotrophin releasing hormone-treated and control cows. The plasma progesterone concentrations on day 8 postbreeding were significantly higher (p less than 0.005) and the change in progesterone concentrations between days 4 and 8 were significantly greater (p less than 0.002) in pregnant cows compared to nonpregnant cows.     

Corpus luteum activity,fertility, and adrenal cortex response in lactating carora cows during rainy and dry seasons in the tropics of Venezuela

The effects of the rainy (RS) and the dry season (DS) on fertility, corpus luteum activity, and adrenal cortex response relationships were evaluated after first service (49 ± 6 d postpartum) in Carora cows, a dairy cattle of Venezuela raised in tropical conditions. Cows (n = 84 in RS and n = 98 in DS) were kept semistabled, had two or three calvings, body condition score 3.5 on a 5-point scale, and similar milk yield (2450 ± 560 kg of milk during the previous lactation). Cows were grouped retrospectively according to pregnancy status. A split-plot model with repeated measures over Days 5, 7, 10, 14, and 15 after insemination was used to analyze the effects of season, pregnancy status, and their interaction involving the day on: 1) serum concentrations of progesterone in four treatments: RS pregnant (n = 26), RS nonpregnant (n = 24), DS pregnant (n = 24), and DS nonpregnant (n = 20) cows; 2) serum concentrations of cortisol at Days 0, 10, 14, 15, and 16 postservice in the previous treatments (n = 9, 7, 6, and 8, respectively); and 3) concentrations of cortisol after 0.1 mg of adrenocorticotropin in these last four groups of cows at Day 14 postin-semination. Breeding during the DS decreased (P < 0.05) conception rate to first service and increased (P < 0.01) days in service. In addition, the DS decreased (P < 0.05) the percentages of cows with normal interestrous interval (20–22 d), expression of estrus, and (P < 0.01) luteal phase progesterone; but DS increased (P < 0.05) percentages of short and long estrous cycles, anovulatory estrus, and repeat breeding rate. Mean serum concentration of progesterone was lower (P < 0.05) at Days 10, 14, and 15 in DS nonpregnant than in DS pregnant cows, and lower during luteal phase (P < 0.05) in DS nonpregnant than RS nonpregnant cows. Serum cortisol concentration was greater (P < 0.05) at Days 10, 14, and 16 in DS nonpregnant than DS pregnant cows. A significant (P < 0.05) negative correlation (r = −0.78) between serum concentrations of progesterone and cortisol was found within DS nonpregnant cows. Concentrations of cortisol after adrenocorticotropin were greater (P < 0.05) in DS nonpregnant cows than in other groups. These results indicate that elevated concentrations of cortisol associated with the DS may decrease progesterone secreted by the corpus luteum and therefore mediate the negative effect of the DS on fertility.     

泌乳期中国荷斯坦牛转化孕酮浓度的影响因素分析及其表型预测

【目的】本研究基于试验测定的中国荷斯坦牛血液和乳汁中的孕酮浓度表型，探究了影响中国荷斯坦牛体内孕酮分泌的环境因素、孕酮浓度的变化趋势、孕酮浓度与乳成分的关联程度以及血乳中孕酮浓度的预测方法。【方法】试验于2021年8月在北京、山东两个牧场采集不同胎次、泌乳阶段和妊娠状态的中国荷斯坦泌乳牛的奶样、尾根血样，测定孕酮浓度，最终获得402条乳汁孕酮浓度和298条血液孕酮浓度表型用于数据分析。对孕酮浓度进行数据转化使其近似服从正态分布后，采用固定效应模型探究胎次、泌乳阶段、妊娠状态、牧场等固定效应对奶牛孕酮表型的影响，运用R语言cor函数计算孕酮与各乳成分间的关联，并利用偏最小二乘法和个体及乳成分信息对孕酮浓度进行预测，以建立孕酮浓度表型高通量获取手段。【结果】妊娠状态对转化乳汁孕酮浓度存在极显著影响（P<0.01），胎次、场效应对转化乳汁孕酮浓度均有显著影响（P<0.05），而转化血液孕酮浓度只受到妊娠状态影响（P<0.01）；全乳固体、乳脂率、乳蛋白率、脂蛋比与转化乳血孕酮浓度均存在显著或极显著的正相关关系（r=0.14~0.37，P<0.05；P<0.01）；基于本试验数据，乳成分与个体信息对转化乳血孕酮浓度的预测准确性不高（R²=0.030~0.17），但如果增加血液或乳汁的转化孕酮浓度对乳汁或血液的转化孕酮浓度进行预测，预测效果则有大幅提升（R²=0.40）。【结论】影响泌乳期中国荷斯坦牛转化孕酮浓度的因素除妊娠状态外，可能还包括饲养条件与胎次。此外转化孕酮浓度与乳脂率、乳蛋白率等乳成分呈极显著相关。基于乳成分信息与转化孕酮的关系，获得了对中国荷斯坦牛乳血转化孕酮浓度预测的可用策略，为今后牧场的繁殖辅助管理、奶牛育种新性状研发以及孕酮浓度的高通量获取等提供了新思路。     

Feed supplementation prevents post-conception decline in milk progesterone concentrations associated with production stress in dairy buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The onset of pregnancy may be associated with hormonal changes and decline in milk yield of buffaloes. To investigate this, forty lactating buffaloes from 1st to 23rd weeks post-conception were selected. The animals were assigned to three treatments: PRT, PRS, NPRT and three milk yielding groups HMY, 66 to 75 l/week, n = 12; MMY, 56 to 65 l/week, n = 16; LMY, 46 to 55 l/week, n = 12). Milk samples were collected on alternate weeks and analyzed with ultrasonic milk analyzer. EIA was used for milk progesterone concentrations. Group means were compared and correlation analysis was conducted. Progesterone concentrations increased in almost similar pattern with the advancing weeks post-conception. The high and low yielder showed greater progesterone concentrations in the supplemented than the animals on traditional ration (P < 0.001). Progesterone concentrations correlated positively with fat (%), negatively with milk yield, protein (%) and lactose (%). Decline in milk yield became drastic when progesterone concentrations rose above 6.44 ng/ml. The pregnant animals on traditional ration exhibited a sharper decline in milk yield with the increasing progesterone concentrations as compared to pregnant animals with supplemented ration. It is concluded that concentrates supplementation induced a raise in progesterone levels. Progesterone concentrations and milk yield showed an inverse relationship.     

Determination of progesterone directly in milk fat as an improved method for fertility control in cattle

A simple and reliable method for the determination of progesterone in milk fat by radioimmunoassay has been developed. Application of the assay under field conditions has shown that all samples diagnosed " questionable" in the former milk progesterone test (MPT) could be characterized as positive or negative. Thus the total figure of animals daignosed correctly rose from 72.3% to 85.8%. The borderlines (ng progesterone / 10 mcl milk fat) set up for this assay were (1) pregnant and (1) nonpregnant (Day 20) and (.3) in estrus and (.3) not in estrus ( Day 0). Within the application for diagnosis of pregnancy, 81.4% were diagnosed correctly as pregnant while 96.3% were diagnosed correctly as not pregnant. The latter figure is somewhat lower than in the MPT (100%), but under the practical conditions the error lies within the range of false diagnosis due to problems with sample identification.