Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: changes with age and effects of cryptorchidism

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.     

Prostaglandin F in the fallopian tube secretion of the ewe

Oviducal secretions were obtained from conscious unrestrained ewes throughout the oestrous cycle via indwelling cannulae and the content of prostaglandin F (PGF) was determined by radioimmunoassay. Levels of PGF of up to 230 ng/ml were found in oviducal fluids obtained from ewes showing regular patterns of secretion and normal cyclical ovarian function as indicated by plasma progesterone measurement. Relatively large day to day fluctuations in content were evident, but there was no consistent relationship between concentration and stage of the oestrous cycle. Concentrations of PGF in excess of 100 ng/ml were common in preparations where autopsy later revealed infection or tissue irritation, and the concentration of PGF invariably exceeded 75 ng/ml when the concentration of protein in the oviducal fluid was abnormally high.     

Elevated Progesterone Levels on the Day of Oocyte Maturation May Affect Top Quality Embryo IVF Cycles

In contrast to the impact of elevated progesterone on endometrial receptivity, the data on whether increased progesterone levels affects the quality of embryos is still limited. This study retrospectively enrolled 4,236 fresh in vitro fertilization (IVF) cycles and sought to determine whether increased progesterone is associated with adverse outcomes with regard to top quality embryos (TQE). The results showed that the TQE rate significantly correlated with progesterone levels on the day of human chorionic gonadotropin (hCG) trigger (P = 0.009). Multivariate linear regression analysis of factors related to the TQE rate, in conventional IVF cycles, showed that the TQE rate was negatively associated with progesterone concentration on the day of hCG (OR was -1.658, 95% CI: -2.806 to -0.510, P = 0.005). When the serum progesterone level was within the interval 2.0–2.5 ng/ml, the TQE rate was significantly lower (P <0.05) than when the progesterone level was < 1.0 ng/ml; similar results were obtained for serum progesterone levels >2.5 ng/ml. Then, we choose a progesterone level at 1.5ng/ml, 2.0 ng/ml and 2.5 ng/ml as cut-off points to verify this result. We found that the TQE rate was significantly different (P <0.05) between serum progesterone levels < 2.0 ng/ml and >2.0 ng/ml. In conclusion, the results of this study clearly demonstrated a negative effect of elevated progesterone levels on the day of hCG trigger, on TQE rate, regardless of the basal FSH, the total gonadotropin, the age of the woman, or the time of ovarian stimulation. These data demonstrate that elevated progesterone levels (>2.0 ng/ml) before oocyte maturation were consistently detrimental to the oocyte.     

Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a qualitative and quantitative milk progesterone test

We compared three methods for diagnosing early pregnancy in cattle: 1) a trans-rectal ultrasound scan of the uterus, 2) a cow-side enzymeimmunoassay (EIA) milk progesterone test 3) a radioimmunoassay (RIA) milk progesterone test. Scanning of the uterus was performed in 148 cows. These cows were not detected in estrus before scanning, which took place between Days 21 and 33 after insemination (AI). A considerable difference was noted between the reliability of the scannings performed at an early stage (Days 21 to 25) and those performed at a later stage (Days 26 to 33). The sensitivity and specificity of the ultrasound examination between Days 21 and 25 were only 44.8% and 82.3%, respectively, but were 97.7% and 87.8% between Days 26 and 33, respectively. Milk samples were collected on the day of AI. (Day 0) and 21 days later. Samples that were positive in the EIA test always contained more than 1 ng/ml progesterone (P4); however, 20% of the negative EIA samples contained also more than 1 ng/ml P4. Only 59% of the animals showing a negative EIA test on Day 0 and a positive test on Day 21, indicating pregnancy, calved, while 16% of the cows with a negative test on Day 0 and Day 21, indicating nonpregnancy, turned out to be pregnant. Of the 82 animals with P4 levels lower than 1 ng/ml on Day 0 and higher than 1 ng/ml on Day 21, only 61.0% calved. All 14 cows with low levels both on Day 0 and Day 21, indicating nonpregnancy, were found to be not pregnant. The influence of both early embryonic death and the accumulation of intrauterine fluids on the accuracy of these tests are discussed.     

Luteinizing Hormone and Gonadal Steroid Levels During the Menstrual Cycle of Orangutans

Scrum luteinizing hormone (LH), progesterone. 17β-estradiol, and testosterone were measured during a single cycle each of five female orangutans, and urinary LH was measured in four of those cycles. Midcycle peaks in LH and luteal phase elevations in progesterone (5.7–13.8 ng/ml) suggested that the cycles were ovulatory. 17β-Estradiol was elevated at midcycle (163–318 pg/ml) and during the luteal phase (56–136 pg/ml) and testosterone was also elevated at midcycle (143–580 pg/ml). These hormone patterns in the orangutans closely resemble those for chimpanzees, gorillas, and human females.     

Uteroglobin and the accumulation of progesterone in the uterine lumen of the rabbit

Summary This study was undertaken to determine whether the influx of progesterone into the uterine lumen of the rabbit, in the preimplantation period, is dependent onuteroglobin (UGL). Rabbits were ovariectomized and, three months later, treated with two defferent doses of progesterone. Purified UGL was injected into one uterine horn and, as a control,immunoglobulin G (IgG) was injected into the other. After four days, the animals were sacrificed their uteri flushed, and the progesterone content of the washes was determined by radioimmunoassay.Animals with the lower serum progesterone level (2.8 ng/ml) had a significantly different uterine horn progesterone content. The hormone accumulation in the horn containing UGL was 2.3 to 7.5 times higher than in the horn containing IgG. Animals with a higher serum progesterone level (7.2 ng/ml) showed no differences. The hormone content was equally high in both horns, presumably due to the synthesis of endogenous UGL being reactivated by the hormone treatment.The validity of these experiments as models for the events during early pregnancy and the physiological role of progesterone available inside the uterus are discussed.     

A triple-X female with long arm deletion of one of the X-chromosomes associated with primary amenorrhoea: 47,XX, +del(X) (q27.3)

A 25-year-old phenotypic female with primary amenorrhoea was referred for chromosomal analysis. Earlier she had undergone hormonal therapy but showed no response. The secondary sex characters were of female type, with poor breast development. Laparoscopic findings revealed the presence of a very small uterus; the right ovary was found to be undeveloped and the left was absent. Cytogenetic study revealed a case of triple-X with deletion of the terminal region of the long arm of one of the X chromosomes [Xq27.3]. Among the 100 buccal mucosa cells analysed, 30 cells showed double Barr bodies. Hormonal studies using RIA technique revealed normal levels of prolactin (9.1 ng/ml), a high level of FSH and LH (135 and 61 mIU/ml) and low levels of estradiol and progesterone (12 pg/ml and 0.20 ng/ml respectively). To our knowledge, this may be the first report of a triple X with deletion of the X chromosome associated with primary amenorrhoea.     

Influence of season and reproductive status on peripheral plasma progesterone levels in the lactating bovine

Data collected monthly for one calendar year from Holstein cows lactating under Louisiana ambient climatic conditions comprised a total of 264 cowmonths. The year was divided into seasons of cool, intermediate, and hot temperatures. A highly significant (P<0.01) relationship between plasma progesterone and animal age (r=–0.57) was observed. Plasma progesterone concentrations in the hot season (4.6 ng/ ml) were significantly (P<0.01) higher than concentrations in the cool and intermediate seasons (3.4 and 3.8 ng/ ml, respectively). Plasma cortisol concentrations were lower (P<0.01) in the hot season than in the cool and intermediate seasons and suggested the adrenal cortex did not contribute to the increase in progesterone concentrations which occured in the hot season. A significant (P<0.01) positive correlation (r=0.45) between cortisol and progesterone was observed. Reproductive status did not have a statistically significant effect on progesterone levels and the affect of season on progesterone concentrations was consistant across all reproductive status. Plasma progesterone levels in the anestrus animals (3.9 ng/ ml) suggested progesterone secretion was responsible for their failure to cycle. Similar progesterone levels were observed in normal (3.5 ng/ ml) and repeat breeders (3.6 ng/ ml).Presented at the Seventh International Biometeorological Congress, 17–23 August 1976, College Park, Maryland, USA.     

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation,activity and steroidogenesis of granulosa cells during human ovarian cortical culture

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.     

Norgestomet- and oestradiol valerate-induced luteolysis is dependent upon the uterus

Beef heifers were assigned to three groups: (1) untreated controls (n=4), (2) Syncro-Mate B(R) (SMB)-treated (n=5), and (3) hysterectomized and SMB-treated (n=4). SMB was administered 8 or 9 days after oestrus, approximately 30 days after hysterectomy. This study was conducted to determine if the uterus was necessary for SMB to induce luteolysis. SMB induced premature luteolysis as only 20% of the intact SMB-treated heifers had >/=0.75 ng/ml of progesterone 7 days after the time of SMB treatment, compared to all (100%) of the untreated heifers (p<0.05). By 9 days after the time of SMB treatment, 25% of the untreated heifers and none (0%) of the intact SMB-treated heifers had >/=0.75 ng/ml of progesterone; however, all (100%) of the hysterectomized SMB-treated heifers had >/=0.75 ng/ml of progesterone (p<0.05). Therefore, SMB-induced luteolysis required the involvement of the uterus. The luteolysin, prostaglandin F(2alpha), is probably the secretion from the uterus that mediates the SMB-induced luteolysis. SMB treatment, however, required 7-8 days to induce luteolysis.     

The ovulatory and LH responses to the male effect in dominant and subordinate goats

The objective of this study was to evaluate the LH and ovulatory response of dominant and subordinate dairy does following the introduction of males. A behavioral study was carried out to determine the individual success index (SI) of 35 anestrous does according to their ability to dominate other females. The 8 highest-ranking (highest SI) and the 8 lowest-ranking does (lowest SI) in terms of dominance were separated from the rest of the herd and placed together in the same pen, where a male was later introduced. Blood samples for plasma LH determinations were obtained from the 16 females at 30 min intervals from 0 to 6, 12 to 18 and 33 to 39 h after the introduction of the male, using an intravenous catheter. After day 8, plasma progesterone was also measured daily in order to determine the occurrence of ovulation. None of the does showed LH pulses during the sampling period prior to the introduction of the male. The dominant does showed significantly (p < 0.05) more LH pulses (2.0 ± 0.18 ng/ml vs. 1.2 ± 0.25 ng/ml) and a higher mean plasma LH concentration (0.25 ± 0.03 ng/ml vs. 0.14 ± 0.03 ng/ml) than the subordinate goats during the first 6 h of exposure to the male. There were no differences in LH pulsatility or concentrations at other times. The frequency of goats that ovulated tended to be greater in high-ranked than low-ranked does (87% vs. 37%). It could be concluded that the immediate LH response to the presence of the male (number of LH pulses) is higher in the dominant, compared to the subordinate goats, and this is associated with a higher number of dominant goats ovulating in response to the male effect.     

Interovarian relationship in the secretion of progesterone during the luteal phase of the capuchin monkey (Cebus apella)

In basal conditions, progesterone concentrations were similar in the ovarian veins of the ovary +CL (3211 +/- 526 ng/ml) and the ovary -CL (3165 +/- 554 ng/ml), but after blocking the blood flow between the ovary +CL and the uterus, the progesterone values in the vein draining the ovary -CL decreased to 1218 +/- 394 ng/ml (P less than 0.01). When [3H]progesterone was injected in the ovary +CL, the radioactivity appeared earlier and more concentrated in the vein draining the ovary -CL (30 sec, 0.53% of injected dose) than in the femoral vein (150 sec, 0.08% of injected dose). Removal of the ovary +CL was followed by a brief maintenance of peripheral progesterone within luteal-phase levels. The in-vitro progesterone production by a suspension of cells isolated from the corpus luteum was 47.5 +/- 12.8 ng/ml/2 h, whereas luteal-like cells isolated from the ovary -CL secreted 14.3 +/- 6.0 ng/ml/2 h (P less than 0.01) into the medium. We therefore suggest that the symmetrical and high secretion rate of progesterone by the ovaries of the capuchin monkey indicates a between-ovary communication system, and that the luteal-like tissue of the ovary -CL can produce relatively large amounts of progesterone.     

Effect on pregnancy and plasma progesterone of exogenous steroid maintenance in ovariectomised pregnant rabbits

Pregnancy was maintained in ovariectomised does with 1 to 4 mg/day of exogenous progesterone with or without 0.2 μg/day of estradiol. Progesterone doses were designed to give similar plasma progesterone levels in treated groups to those found in normal pregnancy, and were measured and compared with normals since this comparison does not appear to have been published previously. In normal pregnancy mean progesterone levels reached 10 ng/ml on day 7 and then plateaued at 14 ng/ml between day 10 and 20 before falling to very low levels at parturition. In treated groups progesterone levels reached 10 ng/ml on day 6 and remained between 10 and 13 ng/ml until day 20 before declining. The difference between treated and control plasma progesterone plateau levels was tested using the t-test and was found not to be significant. The differences in progesterone levels between treated groups with or without estradiol, whether pregnant or not, were also not significant. Mean litter sizes (alive or dead) were not significantly different. However, fetal viability in the group maintained on progesterone alone was significantly lower than in the normal controls.     

Evaluation of different techniques for pregnancy diagnosis in sheep

Fifty Corriedale ewes were used in this study to evaluate pregnancy diagnosis in sheep. Ewes were bred under a pen mating system and pregnancy diagnosis was initiated from day 15 post-mating, applying the diagnostic techniques of trans-abdominal real-time B-mode ultrasonography, Preg-alert (A-mode ultrasonography), the Doppler ultrasonic fetal pulse detector or the plasma progesterone concentration assay (EIA). These tests were repeated fortnightly on all the ewes until the onset of lambing. The accuracy of trans-abdominal real-time B-mode ultrasonography (68%) at days 15–30 of pregnancy increased to 100% by days 61–75 and remained constant until lambing. The accuracy of the Preg-alert (56%) diagnosis at days 31–45 increased to 94% by days 91–105 of gestation and then decreased to 82% from days 136 of gestation to lambing. The accuracy of both the Doppler ultrasound (56%) at days 31–45 and plasma progesterone assay (98%) at days 15–30 of gestation increased to 100% at days 76–90 and 46–60 of gestation, respectively and remained constant until parturition. The mean plasma progesterone concentration at days 0–6 (1.41 ± 0.21 ng/ml) increased to 4.0 ± 0.87 ng/ml at days 16–30 (days 18.23 ± 0.78) post-mating. Animals returning to estrus recorded less than 1 ng/ml at days 18.23 ± 0.78 post-mating. The accuracy of both the B-mode ultrasonic technique (78%) and plasma progesterone assay (98%) was significantly higher (P < 0.05) than the accuracy obtained with the A-mode and Doppler ultrasound (both 56%) at days 31–45 of gestation. The study concluded that real-time B-mode ultrasonography is the earliest, most accurate, safest, fastest and most economical method of pregnancy diagnosis in sheep at farm level. The A-mode and Doppler methods can also be used under field conditions, where sophisticated laboratory facilities are not available. Plasma progesterone assays (EIA) can be used as a means of early pregnancy diagnosis in organized sheep farms with fair accuracy.     

Regional brain and sex differences in the plasma progesterone concentration of sheep

Progesterone concentration was measured in specific brain regions of the ram and anestrous ewe to provide reference values for future studies to investigate if local CNS lesions resulting from neurodegenerative diseases of sheep are associated with progesterone loss. Using radioimmunoassay, plasma progesterone was recorded throughout all brain regions assayed. No significant differences were found between the ewe and ram for any brain regions. There were however, significant differences between the different regions of an individual brain (P < 0.05). Plasma progesterone concentration for the highest to the lowest value recorded was as follows: 2.93 ± 0.85 and 2.77 ± 0.51 ng/g in the frontal cortex; 2.33 ± 0.67 and 2.25 ± 0.48 ng/g in the parietal cortex; 1.32 ± 0.36 and 1.29 ± 0.35 ng/g in the temporal cortex; 1.25 ± 0.32 and 1.25 ± 0.31 ng/g in the occipital cortex; 1.24 ± 0.30 and 1.23 ± 0.31 ng/g in the corpus callosum; 1.16 ± 0.30 and 1.21 ± 0.38 ng/g in the cerebellum; 1.09 ± 0.30 and 1.12 ± 0.39 ng/g in the medulla oblongata of the ewe and ram, respectively. Plasma progesterone concentration in the ewe (0.28 ± 0.06 ng/ml) was significantly higher than that in the ram (0.10 ± 0.03 ng/ml) (P < 0.001). Furthermore, plasma progesterone concentration in all sheep was several times lower than that of any regions of the brain. The results indicate that the sheep brain accumulates progesterone in significant levels, which may be independent of the circulating progesterone. The brain progesterone concentration in CNS regions assayed was similar for the ram and anestrous ewe. Neurodegenerative processes in visna, border disease and enzootic ataxia should be questioned in further studies if they are associated with local progesterone loss.     

Cigarette smoking and serum soluble Fas levels: Findings from the JACC study

Cigarette smoking enhances low-grade systemic inflammation in the lung and other organs. Activated immune cells play an important role at early and late stages of inflammation, and in recent years, soluble Fas (sFas), an isoform of death molecule Fas, was found to interfere with the apoptotic pathways of these activated immune cells. The aim of this study was to confirm the association between cigarette smoking and sFas levels in healthy male subjects. We measured serum sFas levels of 4415 male subjects selected as controls for a nested case-control study within the large-scale cohort study conducted in Japan, called the JACC Study. Smoking status at baseline was evaluated by a self-administered questionnaire. Least square means of sFas according to smoking status and numbers of cigarettes smoked per day among smokers were calculated and adjusted for possible confounding factors. Mean sFas levels showed an increasing trend across never smokers, past smokers and current smokers, as 2.21 (95% CI: 2.14–2.27) ng/ml, 2.29 (2.22–2.36) ng/ml, and 2.36 (2.30–2.43) ng/ml, respectively. However, no dose-response relationship was observed between the number of cigarettes smoked per day and sFas levels among smokers.     

Effect of time and temperature on bovine serum and plasma progesterone concentration

Whole blood, with and without anticoagulant, from 5 pregnant cows was incubated at 40°C for 0 (30 minutes after collection), 6 and 24 hours (hr) before the blood was centrifuged and the plasma or serum was frozen for later progesterone assay. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 1.7 ng/ml at 6 hr (P < 0.01) and to 2.8 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 3.9 ng/ml at 6 hr (P < 0.01) and to 4.4 ng/ml at 24 hr (P < 0.01). Whole blood samples with and without EDTA were also incubated at 4°C for 24 hr. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 4.2 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 4.7 ng/ml at 24 hr (P < 0.01). The incubation time and temperature of whole blood, from collection of blood to the separation of serum or plasma, significantly affects assayable concentration of progesterone.     

Production of steroids by in vitro superfusion from adrenals of the Mongolian gerbil (Meriones unguiculatus): effect of acute stress

1. The output of steroids by in vitro superfusion from adrenals of normal gerbils was studied; the glands secreted the following amounts of steroids (ng/animal/hr): 272 (glucocorticosteroids), 60 (aldosterone), 5.0 (progesterone), 1.0 (androstenedione) and 1.0 (testosterone). 2. Glucocorticosteroid and progesterone output from superfused glands of animals stressed by exposure to a novel environment (a) on concentrated ether vapor (b) was significantly higher than that of control animals (glucocorticosteroids: a: 3-9 min, b: 3-30 min after start of superfusion; progesterone: a, b: 3 min after start of superfusion). 3. Aldosterone output was not affected by the stressors applied. 4. Glucocorticosteroid plasma levels of 204 ng/ml were found in control animals. Exposure to a novel environment or concentrated ether vapor resulted in significantly elevated glucocorticosteroid concentrations (511 ng/ml and 760 ng/ml, respectively). 5. Neither testosterone nor progesterone plasma levels were changed by these stressors.     

Disorder of sexual development in a Yorkshire terrier (78, XY; SRY-positive)

A 9-month-old Yorkshire terrier was admitted to the clinic because of abnormal sexual behaviour and clitoral hypertrophy. External examination confirmed standard development of caudal genital organs: vagina, vulva and cervix uteri. Serum profile of gonadotropin hormones 17 β-estradiol (<10.0 pg.ml^?1) and testosterone (9.1 ng.ml^?1) revealed the presence of testicular tissue. A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicles, structures indicating epididymis and rudimentary deferent ducts were resected, along with adherent part of the uterus. Cytogenetic analysis showed a male chromosomal complement 78, XY in all metaphases of the studied Yorkshire terrier dog. The chromosomal constitution was confirmed by fluorescence in situ hybridisation (FISH) with whole-chromosome painting probes specific for chromosomes X and Y, as well as by polymerase chain reaction (PCR) amplification of the 271-bp Y-linked fragment of SRY (the sex-determining region on the Y chromosome) gene. Sequencing of the dog’s SRY gene coding region did not reveal any mutation. To search for potential mutation in the SOX9 gene (Sry-box containing gene 9), which is considered to be one of the key genes involved in the sex determination process, the PCR fragments of exons 1, 2 and 3 originating from the canine patient were sequenced in order to compare with both male and female healthy control dogs. In the analysed regions of the SOX9 gene, no mutation was found.     

How the concentration of insulin affects the development of preantral follicles in goats

This study investigated the effect of adding different insulin concentrations to the culture medium for goat preantral follicle development in vitro. The ovarian fragments were immediately fixed or cultured for 7 days in MEM with insulin (0, 5, 10 ng/ml and 5 or 10 μg/ml). The results showed that, after 7 days of culture, insulin at 10 ng/ml was the best concentration to preserve follicular viability and ultrastructure, resulting in the highest rates of normal follicles. After 7 days, only treatments with 10 ng/ml and 5 μg/ml of insulin increased follicular activation when compared to other concentrations. Regarding follicular and oocyte growth, the presence of 10 ng/ml of insulin promoted a larger diameter than other treatments. In conclusion, this study shows that addition of 10 ng/ml of insulin to the culture medium improved the survival and stimulated growth of goat preantral follicles.