Identification, purification and immunohistochemical detection of the inhibitor from porcine ovarian follicular fluid to compensatory ovarian hypertrophy in mice.

An inhibitor to compensatory ovarian hypertrophy in mice was detected in porcine follicular fluid. Compensatory hypertrophy was inhibited by treatment with 0.6 ml whole follicular fluid, 0.2 ml of the non-dialysable fraction, or a fraction of the fluid salted out by ammonium sulphate at a saturation of 14.5--18.5%. The salted-out fraction was separable into two peaks by Sephadex G-200 column chromatography and the second peak, detectable as a single band by polyacrylamide gel disc electrophoresis, contained all the inhibitory activity. Specific fluorescence of an antiserum to the second peak was demonstrated at the granulosa cells.     

Hormonal dynamics and follicular turnover in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis)

The aim of this study was the investigation of hormonal and ovarian follicular dynamics in prepuberal buffaloes (Bubalus bubalis) bred in Italy. Eleven 5-9-month old buffalo calves ranging in weight from 122 to 270kg, maintained under controlled nutritional and environmental conditions, underwent 50 days of ultrasonographic ovarian follicular monitoring in the months of October-December. Blood sampling for E(2) and FSH determination and ultrasonographic monitoring using a 7.5MHz linear probe and an ALOKA SSD-500 monitor were performed daily. No differences in any of the parameters under study were highlighted when calves were divided into two weight categories (<200 and >200kg) and thus data were pooled. In this study, values are reported as mean+/-S.D. A range of two-six regular follicular waves was reported among calves with an average of 4+/-1.1. Overall interval (days) between wave emergence was 9.9+/-2.8 and largest diameters (mm) of dominant and first subordinate follicles were 8.4+/-1.2 and 4.8+/-0.6, respectively (P<0.05). With the exception of one calf, some minor follicular waves (short waves or SWs; 1.6+/-1), lasting <10 days (6.1+/-1.2) were reported. They were monitored contemporaneously on the ovary contralateral (n=7) or ipsilateral (n=3) to the main follicular wave. Growth rate (mm per day) of dominant follicles (DF) was significantly faster than for corresponding subordinate follicles (SF) and follicles of SWs (1.08+/-0.2 versus 0.79+/-0.1 and 0.83+/-0.1, respectively, P<0.05). The static phase (days) lasted longer in DF compared to SF and SW (5.4+/-1.8 versus 2.4+/-1.2 and 2.6+/-1, respectively, P<0.05). The regressing phase (mm per day) was similar among DF, SF and SW (0.86+/-0.2, 0.94+/-0.2 and 0.84+/-0.1, respectively, P=0.09). Episodic spikes of E(2) and FSH were reported, corresponding to wave development throughout the course of investigation. In conclusion, the majority of buffalo calves displayed a typical pattern of regular follicular development in conjunction with a dynamic trend of ovarian and hypophyseal hormones. Some minor follicle turnover was reported with parallel main follicular waves.     

Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells

The objective was to determine ovarian follicular fluid concentrations of glucose, lactate, and pyruvate in relation to follicle size in buffalo and sheep. The effect of varying concentrations of these substances on in vitro oocyte maturation, oocyte protein content, and granulosa and cumulus cell growth was also investigated. Follicular fluid was aspirated from various sizes of follicles (from ovaries without a dominant follicle) collected from adult, cycling nonpregnant buffalo (Bubalus bubalis) and sheep (Ovis aries) during the breeding season. Overall, mean (+/-S.E.M.) concentrations (mM) were glucose 2.42+/-0.31 and 1.40+/-0.22, lactate 7.56+/-2.61 and 10.42+/-1.64, and pyruvate 0.02+/-0.01 and 0.002+/-0.00, in buffalo and sheep, respectively. In both species, as follicles became larger, concentrations of glucose significantly increased, lactate significantly decreased, but pyruvate was not affected. Oocyte maturation was higher (P<0.05) in medium containing supra-physiological concentrations of either glucose (5 mM), or pyruvate (10 mM) alone, or physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub- or supra-physiological concentrations of glucose, lactate and pyruvate in combination (both species). The protein content of oocytes was not significantly affected by the concentration of glucose, lactate, and pyruvate in the maturation medium. However, growth of granulosa and cumulus cells was higher (P<0.05) in medium containing supra-physiological concentrations of glucose (5 mM) alone, or pyruvate (10 mM) alone, or physiological, or supra-physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub-physiological concentrations of glucose, lactate and pyruvate in combination (both species). In conclusion, concentrations of glucose, pyruvate and lactate in the medium had cell type-specific effects on oocyte maturation, and on growth of granulosa and cumulus cells. Furthermore, glucose and pyruvate were the principal energy sources for oocytes and follicular somatic cells in buffalo and sheep.     

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using SepPak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70 +/- 0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88 +/- 2.49 pg/ml.     

Changes in uterine protein secretion during luteal and follicular phases and detection of phosphatases during luteal phase of estrous cycle in buffaloes (Bubalus bubalis)

Changes in uterine proteins during different reproductive states and their functional significance though known in other species have not been established in buffaloes. An attempt has been made to unravel the changes in composition of buffalo uterine secretion with growth and regression of corpora-lutea during early, mid and late luteal and follicular phase of estrous cycle using gel filtration and electrophoresis techniques. Also the phosphatases activities in luteal phase uterine secretions have been studied. Gel filtration chromatography analysis revealed a protein peak in void volume of the column, the intensity of which was more in all the luteal phase samples than follicular phase samples. Alkaline phosphatase was also found eluted in the void volume. The other three uterus-specific peaks (Peaks V-VII) were detected below 13.7 kd molecular weight. There were at least five peaks of acid phosphatases activity in chromatogram. Silver staining of SDS-PAGE gel detected as many as 40 protein bands in the uterine fluid of which nine proteins were glycoproteins. Molecular weight (MW) comparison revealed the major protein band at 66 kd which could be serum albumin. Comparison of uterine proteins with serum protein bands revealed a 93.5 kd glycoprotein in buffalo serum that did not appear in uterine fluid and at least 11 uterus-specific protein bands (506, 470, 241, 114, 49, 38, 33, 26, 19.2, 16, and 14.3 kd). The 38 and 19.2 kd bands were luteal-stage specific. Intense periodic acid Schiff's (PAS) stained bands in uterine proteins compared to serum indicated glycosylation process in endometrial epithelial cells. The study suggested that buffalo uterine secretion contained mainly serum and several uterus-specific proteins of which few were luteal phase specific. Further study on characterizing the unique or most abundant proteins and defining their role in uterine functions would help to address the cause of low reproduction rate in buffaloes.     

Blood testosterone levels in Italian Mediterranean buffalo bulls managed in two different breeding conditions

In Italian buffalo cows the spontaneous cyclic ovarian activity is mainly high in autumn, while during spring and early summer it is very low. However many farmers separate males from females in the October-February period to obtain births in winter-spring. In order to verify if blood testosterone concentration in adult buffalo bulls is affected by season and by different management of the contact with females, 20 adult buffalo males, bred in central Italy were submitted to monthly blood sampling for 1 year, from September to August. The bulls were kept together with females all the time (group A; n=9) or were held separated from cows from October to February (group B; n=11). The mean (+/-S.E.M.) serum testosterone concentrations were higher in spring and summer than in autumn and winter in group B (2.07+/-0.1 ng/mL versus 0.99+/-0.08 ng/mL, P<0.01) but in group A the seasonal difference was not significant (2.09+/-0.13 versus 1.48+/-0.28). The management of the contact with females affected testosterone values (P<0.01): in the separation period (October-February) the mean serum concentration in group B was lower than in March-September, when the cows were together with the bulls (0.94+/-0.09 ng/mL versus 1.95+/-0.1 ng/mL, P<0.05). This is not true for group A (1.49+/-0.20 ng/mL versus 2.00+/-0.13 ng/mL, NS). It is concluded that contact with females exerted a major stimulus for the testicular androgen secretion in buffalo bulls, even if other seasonal factors (climate, food intake) may affect control of gonadal activity.     

Alteration of compensatory ovarian hypertrophy in the hamster by melatonin

Hamsters were unilaterally ovariectomized on day four of the estrous cycle and the subsequent compensatory ovarian hypertrophy (COH) and compensatory follicular stimulation studied on each of the following nine days. The hypertrophy stabilized at approximately 35% by the end of the second postoperative cycle, with greater ovarian weights observed on the mid-cycle days. Melatonin injections significantly reduced follicular counts when unilateral ovariectomy was performed on either day three or day four of the estrous cycle and necropsy was on the same day of the cycle, four days later.     

Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis)

The growth, selection, regression and ovulation of ovarian follicles was ultrasonically monitored in 30 Murrah buffalo throughout a spontaneous estrous cycle during the breeding season (autumn). Examinations revealed that follicular growth during the estrous cycle occurs in waves; the buffalo showed 1-wave (3.3%, n = 1), 2-wave (63.3%, n = 19) or 3-wave (33.3%, n = 10) follicular growth. The first wave began at 1.00, 1.16 +/-0.50 and 1.10 +/- 0.32 d in buffalo with 1, 2 and 3 waves, respectively (ovulation = Day 0). The second wave appeared at 10.83 +/- 1.09 and 9.30 +/- 1.25 d (P < 0.01) for the 2 and 3 wave cycle animals, respectively. The third wave started at 16.80 +/- 1.22 d. Structural persistence of the first dominant follicle was longer in the 2- than 3-wave cycles (20.67 +/- 1.18 vs 17.90 +/- 3.47 d ; P < 0.05). The duration of the growth and static phases of the first dominant follicle differed between the 2 and 3 wave cycles (P < 0.05), whereas there were no differences in linear growth rates (cm/d). Two and three wave cycles differed (P < 0.05) with respect to the maximum diameter of both the first dominant follicle (1.51 +/- 0.24 vs 1.33 +/- 0.18 cm) and the ovulatory follicles (1.55 +/- 0.16 vs 1.34 +/- 0.13 cm). No relationship was found between dominant follicle development and the presence of either a CL or a previous dominant follicle in either ovary. Two and three wave cycles also differed with respect to the mean length of intervals between ovulation (22.27 +/- 0.89 vs 24.50 +/- 1.88 d; P < 0.01) and the mean length of luteal phases (10.40 +/- 2.11 vs 12.66 +/- 2.91 d; P < 0.05). These results demonstrate that buffalo have estrous cycles with 1, 2 or 3 follicular waves; that 2-wave cycles are the most common; and that the number of waves in a cycle is associated with the luteal phase and with estrous cycle length.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Presence of immunoreactive dynorphin in human cerebrospinal fluid

Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.     

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.     

Biochemical and hormonal composition of follicular cysts in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular fluid biochemical and hormonal changes associated with ovarian follicular cysts in buffalo. Follicular fluid was aspirated from eight cysts and eight preovulatory follicles, and subjected to biochemical and hormonal analyses. Cysts were characterized by a greater (P<0.01) concentration of nitric oxide and lesser concentrations of ascorbic acid and glucose than that of preovulatory follicles (P<0.01 and P<0.05, respectively). Furthermore, follicular cysts had greater concentrations of progesterone (P<0.001), triiodothyronine (T(3)) and cortisol (P<0.05) and lesser concentrations of insulin (P<0.001) than preovulatory follicles. The results indicate follicular cysts in buffalo have an altered biochemical and hormonal composition. The alterations include increases in nitric oxide, progesterone, cortisol and T(3) concentrations with a concurrent reduction in ascorbic acid, insulin and glucose concentrations. The study suggests that greater progesterone concentrations possibly inhibit the onset of LH surge resulting in formation of follicular cysts in buffalo. In addition, it implies the plausible role of intra-ovarian regulators such as nitric oxide, ascorbic acid and insulin in development of the condition.     

Follicle stimulating hormone secretion and compensatory ovarian hypertrophy in prepubertal ewes

A study was conducted to determine the effect of unilateral ovariectomy (ULO) on follicle stimulating hormone (FSH) secretion and compensatory ovarian hypertrophy in prepubertal ewes. Thirty-three ewe lambs were allotted according to age and weight to a control (C) or ULO group. In the C group, a sham ovariectomy was performed on day 0 and both ovaries were removed on day 7. In the ULO group, one ovary was removed on day 0 and the remaining ovary was removed on day 7. Blood samples were collected from the jugular vein via venipuncture at 0, 6, 12 and 24 hours after the time of sham surgery or ULO (day 0). Subsequent samples were collected daily until day 7, and all samples were assayed for FSH and LH. Unilateral ovariectomy increased (P<0.01) ovarian weight and follicular fluid weight; however, lyophilized ovarian weight was similar for both groups. Within the ULO group, removal of the ovary having the largest follicle(s) did not prevent an increase in ovarian weight or follicular fluid weight of the remaining ovary. Unilateral ovariectomy had no effect on the total number of follicles (1 to 6 mm) per ovary; however, the number of large (5 to 6 mm) follicles per ovary was increased (P<0.05) following ULO. By 12 hours after ULO there was a transient increase (P<0.05) in the circulating concentrations of FSH. Circulating concentrations of luteinizing hormone (LH) were either low or undetectable in these prepubertal ewes and no LH response was observed following ULO. These results indicate that compensatory ovarian hypertrophy in ULO prepubertal ewes is accompanied by a transient rise in circulating FSH concentrations.     

Purification, measurement, and tissue distribution of a dansyl-derivatized glycopeptide from low-molecular weight follicle-stimulating hormone-inhibitor-containing fractions of porcine follicular fluid

We have used dansyl chloride (5-dimethylamino-1-naphthalenesulfonyl choloride) to form dansyl derivatives of amine-containing compounds in follicular fluid or highly purified fractions containing a low molecular weight (MW) inhibitor of follicle-stimulating hormone (FSH) binding to receptor (FSH-BI). This approach allowed sensitive detection of the derivatives based on their fluorescent properties. By taking advantage of the hydrophobic nature of the dansyl group, a dansyl derivative (RF = 0.15) identified in low MW FSH-BI preparations was purified from porcine follicular fluid. Based on chromatographic criteria using four different systems (thin-layer chromatography [TLC] and high performance liquid chromatography), the derivatized factor (D15) that was purified appeared to be homogeneous. A direct, chemical assay was developed for quantification of D15 from follicular fluid or tissue extracts. The highest concentration (153 ng/mg) of D15 was found in ovarian tissue of adult rats, lesser amounts were observed in kidney and liver tissues (93 and 62 ng/mg, respectively) and even less in diaphram and heart tissues (5 and 0.5 ng/mg, respectively). High concentrations of D15 were observed in derivatized extracts of tests from immature rats in which approximately twice as much D15 was found in Leydig cells (241 ng/mg) as in seminiferous tubules (136 ng/mg). In porcine ovarian tissue, granulosa cells from large follicles and corpora lutea (69 and 91 ng/mg, respectively) contained at least 4-fold higher concentrations than follicle wall tissue (14 ng/ml). Relative concentrations of D15 material were also determined in pools of bovine follicular fluid previously shown to contain low MW FSH-BI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Native vitellins are modified during ovarian development in the stick insect Carausius morosus (Br.)

Vitellins from ovarian follicles and newly laid eggs of the stick insect Carausius morosus were examined by ion exchange chromatography on a HPLC Mono Q column. Under these conditions, vitellins from newly laid eggs resolved as two distinct peaks, referred to as VtA and VtB, that eluted at 8.5 and 12.0 min, respectively. On native gels, both VtA and VtB separated into two different variant forms (VtA′ and VtA′, VtB′ and VtB′). By two-dimensional gel electrophoresis, VtA′ and VtA′ were shown to contain polypeptides A₁, A₂ and A₃. On the other hand, VtB′ and VtB′ appeared to comprise polypeptides B₁ and B₂ and B₁, A₁, A₂, B₂ and A₃*, respectively. A similar Vt polypeptide composition was also observed by size-exclusion chromatography of vitellins from newly laid eggs. Vitellins from early vitellogenic ovarian follicles resolved into a single chromatographic peak at 7.5 min that coeluted with a major peak from the hemolymph of egg-laying females. Ovarian follicles progressively more advanced in development exhibited a more complex chromatographic profile, consisting of three separate peaks. By two-dimensional gel immunoelectrophoresis, vitellins from ovarian follicles appeared to consist of two closely related, immunologically cross-reacting antigens that gradually shifted apart as ovarian development proceeded to completion. By size-exclusion chromatography, each Vt from ovarian follicles was shown to consist of a unique set of polypeptides different from those listed above. Single ovarian follicles were fractionated into yolk granules and yolk fluid ooplasm and tested by immunoblotting against Mab 12. Under these conditions, VtA variant forms in yolk granules and yolk fluid ooplasm reacted differently. Sections from ovarian follicles in different developmental stages were exposed to Mab 12 and stained with a peroxidase-conjugated, goat anti-mouse antibody. Regardless of the developmental stage attained, staining for peroxidase was restricted to free yolk granules, suggesting that native vitellins in stick insects are structurally modified upon fusion into the yolk fluid ooplasm. Arch. Insect Biochem. Physiol. 36:335–348, 1997. © 1997 Wiley-Liss, Inc.     

Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats (Capra hircus)

Information on the use of buffalo follicular fluid (buFF) in modulation of ovarian functions in farm animals is scanty compared to other species. This is an attempt to investigate the effect of direct administration and active immunization of 30 kDa and above buFF proteins on ovarian functions in goats. Treatment of goats (n = 6) with steroid free 30 kDa and above buFF protein fraction during late-luteal phase for 4 days (days 12 or 13 to days 15 or 16) of the natural cycle, delayed the onset of estrus by 24 h compared to control although the mean duration of estrus was unaffected. A 71% increase (P = 0.06) in mean ovulation number was also observed following treatment. However, the population of large (> or =5 mm diameter) follicle was not affected. The ovarian activity calculated as total of ovulation and large follicles increased (1.6 times) significantly (P = 0.02) in treated animals. Active immunization of goats (n = 5) against these proteins did not affect the onset and duration of estrus. Similarly, the ovulation rate, number of large follicles and the ovarian activity did not differ significantly between immunized and control groups. The study revealed that 30 kDa and above buffalo follicular fluid contains some factor(s) that cause delay in the onset of estrus in goats and increase the ovulation rate. Active immunization against these proteins in goat did not show any effect either on onset, duration of estrus or ovulation rate and large follicle population. Detailed study on these buffalo follicular fluid proteins may help to use them further for modulation of ovarian function in farm animals.     

Identification of insulin-like growth factor-II in human seminal and follicular fluids

Antisera raised in rabbits against synthetic insulin-like growth factor-II (IGF-II) were used to develop a specific radioimmunoassay (RIA) for IGF-II. Affinity purified antibodies showed 6% cross-reactivity with IGF-I but failed to recognize insulin even at 10 micrograms/tube. Utilizing this RIA system, immunoreactive IGF-II was identified in the pooled samples of human follicular fluid and seminal plasma. The acid-ethanol precipitates of human seminal and follicular fluids were chromatographed on Sephadex G-50 column and the IGF-II immunoreactive fractions were subjected to reversed-phase high performance liquid chromatography. It was found that immunoactive IGF-II was eluted in the same location as that of synthetic IGF-II. The data indicate for the first time that human seminal plasma and follicular fluid contain significant amounts of IGF-II.     

Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration

In the separation of the triplet components of neurofilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 m urea and a linearly increasing sodium phosphate concentration from 10 to 400 mm at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partially separated with 6 m urea, and completely separated with 4 m urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurofilament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 m urea at first, and then P 160 and P 68 were separated in the presence of 4 m urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 m.     

SEASONAL CHANGES IN FAVOURABILITY OF CATTLE DUNG IN CENTRAL QUEENSLAND FOR THREE SPECIES OF DUNG-BREEDING INSECTS

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The favourability of cattle dung from a native pasture near Rockhampton, Queensland, as a food source was tested monthly in the laboratory for 2 yr using 3 dung-breeding insects: the buffalo fly, Haematobia irritans exigua De Meijere; the bush fly, Musca vetustissima Walker; and a dung beetle, Euoniticellus intermedius (Reiche). Dung produced by cattle grazing on this pasture during the summer wet season yielded larger flies of both species and more broods from the dung beetle than dung from the same pasture in winter. When reared in summer dung, the buffalo fly almost attained its maximum recorded size but the bush fly and dung beetle reached ca two-thirds maximum recorded size and brood production respectively. Bush flies failed to breed in dung collected for 4 consecutive months in winter each year but survival of buffalo flies showed no seasonal trends.
The early response of the buffalo fly to improving dung quality in late winter/early spring gives it an advantage enabling its populations to increase earlier than those of its competitors, including the dung beetle, E. intermedius.     

Laparoscopic ovum pick-up and in vitro production of sika deer embryos: effect of season and culture conditions

Amongst the 200 deer subspecies worldwide, more than 40 are considered as endangered. In vitro embryo production may represent an efficient way to produce and disseminate offspring from sparse remaining individuals in these species. With a view to establishing a method of in vitro embryo production, we assessed the ovarian response after hormonal stimulation (oFSH), oocyte yield following laporoscopic ovum pick-up (LOPU) and oocyte developmental competence according to seasonal reproductive status in sika deer (Cervus nippon nippon). Twelve adult sika deer hinds were allocated between two groups and submitted weekly to oFSH follicular growth stimulation followed by LOPU. Hinds in Group A (n=6) were treated first during the breeding season (5 weeks), and then during the non-breeding season (3 weeks). Hinds in Group B (n=6) were submitted to similar procedures but in the reverse order (treated first during the non-breeding season). Cumulus-oocytes complexes (COC) recovered from Group B were allowed to mature in vitro for 24 h in TCM-199 medium supplemented with oFSH, goat follicular fluid and 100 microM cysteamine. In vitro fertilization was performed with frozen/thawed semen in SOFaa medium supplemented with 20% estrous sheep serum and presumptive zygotes were cultured in the presence or absence of ovine oviductal epithelial cell monolayer (oOEC) in SOFaa-BSA medium. Mean number of follicles aspirated per hind per session decreased significantly between breeding and non-breeding season in Group A (9.8+/-0.7 versus 3.2+/-0.7, mean+/-S.E.M., respectively, P<0.001) but did not change between the non-breeding and the subsequent breeding season in Group B (5.3+/-0.7 and 5.7+/-0.7, respectively, P>0.05). Irrespective of the season, good quality COC with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Whereas development to the blastocyst stage did not occur in SOF medium alone, high development rates to the blastocyst stage were observed in oOEC co-culture regardless of season (22% and 34% of total oocytes in co-culture during non-breeding and breeding season, respectively).