Effects of exogenous FSH on follicular recruitment in a viviparous lizard Niveoscincus metallicus (Scincidae)

In the viviparous skink Niveoscincus metallicus clutch size appears to be determined before vitellogenesis, and is not altered later by follicular atresia or embryonic loss. This suggests that the number of follicles recruited is determined by the endocrine environment early in the vitellogenic period. Through a series of experiments in which we manipulated gonadotropin concentrations by administering exogenous FSH, we aimed to investigate this hypothesis. Pre-vitellogenic females showed no response to exogenous ovine FSH. In early vitellogenic females, FSH induced follicular recruitment: follicles were enlarged and clutch size increased by recruitment of a second cohort of follicles; some females also ovulated. Females treated with FSH in mid-vitellogenesis had elevated mean plasma estradiol concentrations compared to controls; no follicular recruitment was observed, but most of these animals ovulated. Females treated with a range of doses of FSH in late vitellogenesis ovulated at least one month before natural ovulation, again without recruitment of extra follicles. It appears therefore that in Niveoscincus metallicus exogenous FSH can induce recruitment of additional follicles only if administered during early vitellogenesis. We conclude that in this species clutch size is determined by proximate environmental factors influencing gonadotropin levels early in follicular recruitment, and cannot be increased even if conditions become more favourable once vitellogenesis is established.     

Lipids in yolks and neonates of the viviparous lizard Niveoscincus metallicus

Niveoscincus metallicus is a small viviparous skink which provides a substantial amount of yolk to each of its developing embryos although some organic nutrients are also transferred across the placentae. The total amount of lipid present in the yolk of N. metallicus (37% of dry weight) is very much higher than that in the newborns (19% of dry weight), confirming that the yolk is utilised as an energy source during gestation. Triacylglycerols (TAG), which are storage compounds, are the major lipid resource available to the embryos and are present in relatively large amounts in the yolk of N. metallicus. Polar lipids (PL), which form the structural components of membranes, and sterols (ST), which are involved in the synthesis of hormones and vitamins, are also present in the yolk. The proportions of each of these lipid classes differs markedly between yolks and newborns. This may reflect variations in the role played by each lipid class in the provision of nutrients to, and development of, embryos.     

Effect of exogenous administration of buffalo follicular fluid on follicular development, estrus response and luteal function in anoestrous goats (Capra hircus)

The effect of buffalo follicular fluid (buFF) on follicular development, estrus response and luteal function was investigated in anoestrous does. Treatment with buFF (18 ml/doe) had no significant effect on the number of antral follicles of all class categories during the period of administration. However, after cessation of buFF treatment, the number of total antral follicles increased significantly with time (P < 0.003) as well as due to the treatment × time interaction (P < 0.02), without any influence on follicle size. Injection of buFF also caused a marked increase (P < 0.049) with time in the number of medium-sized follicles at cessation. Approximately 60 and 20% of buFF-treated anoestrous does showed behavioural and silent estrus, respectively, compared to none in the control. The mean interval between cessation of buFF treatment to onset of oestrus and oestrus duration was 67.0 ± 18.5 and 17.0 ± 3.6 h, respectively. Corpus lutea size varied between 4.6 and 5.8 mm with an average diameter of 5.2 ± 0.3 mm. Only 33.3% of does showed serum progesterone levels above 1 ng/ml, while the remainder (66.7%) had below 0.5 ng/ml. Our results indicate that exogenous administration of buFF causes enhanced follicular activity following cessation of treatment, which results in behavioural oestrus and corpus luteum (CL) development in anoestrous does. CL development and its function is, however, inadequate in buFF-treated anoestrous does.     

The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model

The efficacy of a recently engineered single chain recombinant equine follicle stimulating hormone (reFSH) was investigated in estrous cycling mares whose gonadotropins and follicular activity had been suppressed by concurrent treatment with progesterone and estradiol (P&E). Time of estrus was synchronized in 15 estrous cycling mares during the breeding season with prostaglandins F_2α (PGF_2α). The day after ovulation, mares were treated once daily with P&E for 14 days. Mares received a second injection of PGF_2α on day 6 of the synchronized estrous cycle to induce luteolysis. On day 8 post-ovulation mares were randomly assigned to three groups: small dose reFSH-treatment group (0.5 mg reFSH IV, twice daily); large dose reFSH-treatment group (0.85 mg reFSH IV twice daily); control group (saline IV, twice daily). reFSH treatment occurred concurrently with the last week of P&E treatment. After a follicle or cohort of follicles reached 35 mm in diameter, mares were injected with 0.75 mg of recombinant equine luteinizing hormone (reLH) to induce ovulation. Post-treatment ovulation was assessed. Daily blood samples were collected for analysis of FSH, LH, estradiol, progesterone, and inhibin by radioimmunoassay (RIA). On the first day of reFSH/saline treatment, blood samples were collected periodically from 1 h prior to treatment to 6 h post-injection via an indwelling jugular catheter to determine acute changes in FSH concentrations. Monitoring of follicular activity, estrus, and ovulation was performed daily by utilizing a stallion and transrectal ultrasonography.A difference (p ≤ 0.05) between the largest diameter follicle in the reFSH-treatment groups compared to controls occurred on day 14 post-ovulation, the day treatments ended, and the difference continued until day 21 post-ovulation. reFSH-treatment groups had larger (p ≤ 0.05) numbers of 20–29 mm follicles (days 13–18), 30–34 mm follicles (days 15–20) and ≥35 mm follicles (days 16–21) than controls. Mares treated with reFSH, at either dose, took less time (average: 2.95 ± 0.42 days) to develop 2–3 times more pre-ovulatory follicles than control mares (7.8 ± 0.51 days) (p ≤ 0.05). The number of ovulations between treated mares and controls were similar due to a greater incidence of ovulation failure in reFSH-treated mares. During reFSH treatment, concentrations of plasma FSH, inhibin and estradiol were greater (p ≤ 0.05) compared to control concentrations. Plasma LH concentrations in reFSH-treated mares were suppressed and did not exhibit the ovulatory surge of controls (p ≤ 0.05). Plasma progesterone concentrations were not different across groups.These findings demonstrate the specific effects of reFSH to increase number of total follicles including pre-ovulatory follicles in mares with endogenous pituitary gonadotropins and follicular growth suppressed by a regimen of P&E.     

Energy consumption by embryos of a viviparous lizard, Eulamprus tympanum, during development

Energy consumption during development has been measured in many oviparous lizards, but not in viviparous lizards in utero. It has always been assumed that energy consumption by embryos of viviparous lizards during development is similar to that of oviparous species. Estimation of energy consumption of viviparous lizards in vivo are confounded by the possible influence of pregnancy on maternal metabolism. Here we separated maternal and embryonic metabolism in measurements of pregnant Eulamprus tympanum throughout pregnancy. Our data support the hypothesis that the energetic cost of development in viviparous lizards (19.8 kJ g⁻¹) is similar to that in oviparous lizards (mean 16.2 kJ g⁻¹), at least for a species with a simple placenta. An increase in maternal metabolism of 29% above that for non-pregnant E. tympanum goes to maintain pregnancy, and represents an important component of the reproductive effort in E. tympanum.     

Interembryonic regions of the uterus of the viviparous lizard Mabuya brachypoda (Squamata: Scincidae)

Analysis of the structure and physiology of the uterine incubation chambers of viviparous squamates has provided insight concerning adaptations for gestation. However, the literature addressing the biology of the interembryonic regions of the uterus is very limited, presumably because it has been assumed that this area has little role in the development and support of embryos in viviparous squamates. This study was undertaken to examine the histology of the interembryonic regions of Mabuya brachypoda, a viviparous lizard with microlecithal ova and consequently substantial matrotrophic activity. The incubation chambers are oval, distended zones of the uterus, adjacent to the interembryonic regions. The wall of the interembryonic regions includes: mucosa, formed by a cuboidal or columnar epithelium with ciliated and nonciliated cells, and a lamina propria of vascularized connective tissue containing abundant acinar glands; myometrial smooth muscle consisting of inner circular and outer longitudinal layers; and serosa. The segment of the interembryonic region adjacent to the incubation chamber forms a transitional segment that displays folds of the mucosa that protrude into the uterine lumen. The limit of the incubation chamber is well defined by the long mucosal folds of the transitional segment. Long and thin extensions of extraembryonic membranes are present in the lumen of the transitional segment, outside of the incubation chamber region. The presence of abundant uterine glands and extraembryonic membranes in the interembryonic regions during gestation suggests uterine secretory activity and histotrophic transfer of nutrients to embryos in these regions.     

Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH)

Traditionally, mares are put under artificial lights to advance the first ovulation of the year. The aim of the present study was to determine the efficacy of recombinant equine FSH (reFSH) in stimulating follicular development and advancing the first ovulation of the year in seasonally anestrous mares compared with anestrous mares given a placebo. Both groups of mares were housed under ambient light conditions. Sixty deep anestrous mares of light horse breeds (follicular diameters ≤20 mm in diameter and progesterone <1 ng/mL) were maintained under a natural photoperiod at three different sites: University of California, Davis, Colorado State University, and University of Kentucky Gluck Centre. Twenty mares at each site were randomly allocated to receive either 0.65 mg of reFSH (group A: treatment; n = 10) or a placebo (group B: control; n = 10) twice daily by im beginning on January 31. Treatment continued until one or more preovulatory follicles developed or up to a maximum of 15 days. Randomized treatments were blinded. Follicular development was closely monitored by transrectal ultrasonography. When the largest follicle reached ≥35 mm in diameter, reFSH treatment was discontinued and an injection of 2500 international units of hCG was administered iv 36 hours later to induce ovulation. Jugular blood samples were collected daily from all mares at University of California, Davis, and processed for LH, FSH, progesterone, estradiol-17β, and immunoreactive-inhibin by RIA. All 30 mares receiving reFSH (group A) developed follicles ≥35 mm within 7.4 ± 1.6 days of treatment. Twenty-three of the 30 reFSH-treated mares (group A) ovulated within 72 hours after hCG administration. In contrast, mares in group B (placebo, control) did not exhibit significant follicular development and none ovulated within the 15-day observation period. Mares in group A had significantly higher plasma levels of FSH, estradiol-17β, and immunoreactive-inhibin during treatment but did not exhibit a preovulatory LH surge. Mares administered reFSH returned to anestrus and spontaneously ovulated at a similar calendar date as control mares. These data indicate that reFSH was effective in stimulating the development of ovarian follicles and advancing the first ovulation of the year in seasonally anestrous mares under ambient lights but was not successful in inducing continued cyclicity.     

Uterine epithelial morphology and progesterone receptors in a mifepristone-treated viviparous lizard Pseudemoia entrecasteauxii (Squamata: Scincidae) during gestation

Structural and functional changes to the uterus associated with maintenance of pregnancy are controlled primarily by steroid hormones such as progesterone. We tested the hypothesis that progesterone regulates uterine structural changes during pregnancy in the viviparous skink, Pseudemoia entrecasteauxii, by treating pregnant females with the progesterone receptor antagonist mifepristone at different stages of pregnancy. Expression and distribution of progesterone receptor was determined using Western blot and immunohistochemistry. During early pregnancy, mifepristone treatment resulted in altered uterine epithelial cell surface morphology and high embryo mortality, but did not affect females at mid and late stages of pregnancy. Females treated with mifepristone in early pregnancy exhibited abnormal uterine epithelial cell morphology such as lateral blebbing and presence of wide gaps between cells indicating loss of intercellular attachment. Chorioallantoic membranes of the embryo were not affected by mifepristone treatment. Two isoforms (55 kDa and 100 kDa) of progesterone receptor were identified using immunoblots and both isoforms were localized to the nucleus of uterine epithelial cells. The 55 kDa isoform was expressed throughout pregnancy, whereas the 100 kDa isoform was expressed during mid and especially late pregnancy. In P. entrecasteauxii, mifepristone may prevent successful embryo attachment in early pregnancy through its effects on uterine epithelial cells but may have little effect on pregnancy once the maternal-embryo structural relationship is established.     

Effects of ovariectomy and estrogen replacement on attractivity and receptivity in the red-sided garter snake (Thamnophis sirtalis parietalis)

Activation of courtship behavior in male red-sided garter snakes, Thamnophis sirtalis parietalis, is independent of the presence of sex steroids. The only consistent treatment that stimulates courtship behavior in males is prolonged exposure to low temperature followed by subsequent warming, mimicking the emergence from hibernation. We investigated whether attractivity and receptivity in female red-sided garter snakes is similarly steroid independent.Female red-sided garter snakes are attractive when they emerge from hibernation and are courted by males; most mate within an hour of emergence. In a series of experiments, groups of females were either ovariectomized (OVEX) in the late spring, fall or while in hibernation. They were tested for attractivity and receptivity upon emergence from hibernation. Females OVEX in the spring were unattractive whereas those OVEX in fall or while in hibernation were attractive. Thus, attractivity appears determined the year before emergence and is dependent on the presence of the ovaries. All OVEX females were unreceptive upon emergence. OVEX females were also given replacement estradiol (E) treatment (either in Silastic capsules or single injections) at various points of their annual cycle. The only treatment that resulted in reinstating receptivity in OVEX females was the injection of E (20 g) one hour prior to emergence. The effectiveness of E in reinstating receptivity was time dependent: the longer the period between emergence and injection, the less effective the same dosage was in stimulating receptive behavior.These experiments suggest that sexual behavior in female red-sided garter snakes is, unlike males, dependent on the presence sex steroid hormones. Although E is naturally at its lowest seasonal level upon emergence, the concentration is sufficient to stimulate receptivity. However, it appears that temperature regulates a time-limited window of sensitivity to E.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

Influence of somatic cell count, body condition and lameness on follicular growth and ovulation in dairy cows

The objective of this study was to investigate the effect of somatic cell count (SCC), body condition score (BCS) or lameness score on ovarian follicular growth and ovulation in dairy cows. Seventy four animals 30-80 days post-partum were monitored for all three conditions before synchronization of ovarian follicular phases by administration of gonadotrophin releasing hormone (GnRH) followed seven days later with prostaglandin F2alpha (PG). Ultrasonography of both ovaries twice daily throughout the follicular phase revealed that fewer animals with combined high SCC and lameness (4/9) ovulated compared to healthy animals (19/21; P = 0.006) or animals with only high SCC (11/11; P = 0.004) or only lameness (21/27; P = 0.06). Overall, regardless of the presence of other concurrent conditions, fewer lame cows ovulated than Non Lame animals (30/42 and 30/32; P = 0.015). Mean follicular growth and maximum follicular diameter were unaffected by any of the three conditions. However, dominant follicle growth and maximum diameter were greater in the 60 animals that ovulated compared to the 14 that did not; 1.83 ± 0.16 versus 0.96 ± 0.26 mm/day (P = 0.014) and 19.4 ± 0.4 versus 16.4 ± 1.2 mm (P = 0.003), respectively. In conclusion, lameness reduced the proportion of cows that ovulated and the synergistic effect of high SCC and lameness reduced that proportion further. However, follicular growth and maximum follicular diameter were unaffected by high SCC, low BCS or lameness.     

Effects of exogenous progesterone and cloprostenol on ovarian follicular development and first ovulation in prepubertal heifers

The objective of this study was to determine the effects of progesterone and cloprostenol (a PGF_2α analogue) on ovarian follicular development and ovulation in prepubertal heifers. In Experiment 1, crossbred Hereford heifers (Bos taurus; 10 to 12 mo old, 255 to 320 kg) were assigned randomly to three groups and given (1) an intravaginal progesterone-releasing insert (CIDR; P group, n = 13); (2) a CIDR plus 500 μg cloprostenol im (PGF_2α analogue) at CIDR removal (PPG group, n = 11); or (3) no treatment (control group, n = 14). The CIDR inserts were removed 5 d after follicular wave emergence. Progesterone-treated heifers (P and PPG groups) had a larger dominant follicle than that of the control group (P = 0.01). The percentage ovulating was highest in the PPG group (8 of 11, 73%), intermediate in the P group (4 of 13, 31%), and lowest in the control group (1 of 14, 7%; P < 0.02). In Experiment 2, 16 heifers (14 to 16 mo old, 300 to 330 kg) were designated to have follicular wave emergence synchronized with either a CIDR and 1 mg estradiol benzoate im (EP group, n = 8) on Day 0 (beginning of experiment) or by transvaginal ultrasound-guided ablation of all follicles ≥5 mm on Day 3 (FA group, n = 8). On Day 7, CIDRs were removed in the EP group, and all heifers received 500 μg cloprostenol im. Ovulation was detected in 6 of 8 heifers (75%) in both groups. In summary, the use of PGF_2α with or without exogenous progesterone treatment increased the percentage ovulating in heifers close to spontaneous puberty.     

Immediate and carryover effects of Gram-negative and Gram-positive toxin-induced mastitis on follicular function in dairy cows

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF_2α on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.     

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.     

Reproductive patterns in seventeen species of warmwater fishes in a Missouri River reservoir

Synopsis The timing of ovarian maturation and spawning of 17 warmwater fish species in Lake Oahe (South and North Dakota) was estimated from changes in the mean ovary indices (ratios of ovary weight to fish length). The onset of vitellogenesis varied within species (up to 2 months). Maturation of the ova took from 7.5 to 10 months, depending on species. Annual variations in the mean date of peak spawning of individual species during 1964–1971 were usually less than a week. There was little overlap of the annual mean peak spawning dates of the 17 species, and an established sequence of spawning among species was shown. A relatively high incidence of atresia in the shovelnose sturgeon, northern pike, and carp indicated that these species had apparently not yet adapted to the altered and variable spawning conditions in this reservoir. Regularity of spawning would seem to provide the best chance for spawning success in variable environments such as Lake Oahe.     

Interrelationships among plasma progesterone concentrations, luteal anatomy and function, and placental ontogeny during gestation in a viviparous lizard (Niveoscincus metallicus: Scincidae)

Plasma progesterone concentrations were measured at six stages of gestation in the viviparous lizard Niveoscincus metallicus. Anatomical and functional parameters of luteal activity were also investigated. The diameter of the corpus luteum (CL) decreased gradually though gestation, as did the diameter of the luteal cells. Major degenerative changes were observed in CLs post-partum. Plasma progesterone concentrations were basal both prior to, and just after, ovulation; a rapid increase occurred in early gestation. Plasma progesterone concentrations remained elevated until late gestation, but fell some 2 weeks before parturition. In vitro production of progesterone was greater in CLs in mid- than in late-gestation, and the addition of prostaglandin F(2alpha) to the incubation medium had no effect on progesterone production. Non-luteal ovarian tissue and adrenals produced progesterone, but at approximately one-tenth the rate of production by CLs. Temporal correlations between the plasma progesterone profile and stages of placental development were also assessed. The rise in plasma progesterone concentrations occurs before differentiation of the chorioallantoic placenta, but progesterone is still high when it degenerates. We conclude that the CLs are the major source of gestational progesterone in N. metallicus.