Ovaries and testes were collected throughout the year from domestic cats spayed and neutered at local veterinary clinics. Fresh oocytes recovered from minced ovaries were subjected to in vitro maturation and then stained to determine stage of maturation or were inseminated with conspecific sperm. The cauda and corpus regions of each epididymis were dissected into pieces and placed in medium; 30 min later, the epididymal tissue was removed, the medium centrifuged, and the sperm pellet resuspended. Samples were assessed for total sperm count and sperm motility traits, morphology, acrosomal integrity, and ability to penetrate cat oocytes in vitro. Fewer excellent (grade I) oocytes were recovered per ovarian pair during September-November (mean +/- SEM, 19.2 +/- 2.1%) than during January-July (36.8 +/- 3.6%, p < 0.05), while the remaining months had intermediate percentages of grade I oocytes (p > 0.05). A high percentage of oocytes recovered from November-April completed nuclear maturation (64.3 +/- 6.8%), which was different (p < 0.05) from the values for May-July (32.2 +/- 3.8%) and August-October (10.4 +/- 2.9%). Percentage of oocytes with bound sperm was lowest (p < 0.01) in September and October (32.0 +/- 3.1%) compared to February and March (91.4 +/- 1.7%). Percentage of oocytes with sperm within the perivitelline space was highest (p < 0.05) in May-August (33.8 +/- 4.6%) compared to all other months. In contrast, the period of highest (p < 0.01) fertilization (i.e., >/= 4-cell embryo formation) was March-April (51.7 +/- 3.1%) as compared to May-July (17.2 +/- 1.8%) or November-January (12.4 +/- 2.6%). Negligible numbers of oocytes recovered during August-October developed beyond the 2-cell stage (1.1 +/- 0.3%). Blastocyst development from cleaved embryos was highest during February-April (44.3 +/- 2.3%) and lowest during August-October (0.6 +/- 0.1%; p < 0.01). Sperm recovered from the epididymides throughout the year did not differ (p > 0.05) in concentration or in any of the motility, structural, or functional variables evaluated. In summary, cat oocyte nuclear maturation in vitro is depressed during August-October, and the ability to form cleaved embryos remains low even when the capacity to achieve nuclear maturation is relatively high (November-January and May-July). In contrast, male cats are capable of consistently producing viable, progressively motile sperm throughout the year.

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.

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Cryopreservation is an important conservation tool, which may help reef-building coral survive. However, scaling-up from small, laboratory-sized experiments to higher-throughput restoration is a major challenge. To be an effective restoration tool, the cryopreservation methods and husbandry to produce new offspring must be defined. This study examined small and larger-scale in vitro reproduction and settlement for Acropora tenuis and Acropora millepora and found that: 1) cryopreservation of coral sperm reduced sperm motility and fertilization success in half, thus fresh sperm, capable of becoming highly motile, is key; 2) the sperm-to-egg ratio and the concentration of the cryoprotectant treatments affected fertilization success in small- and larger-scale reproduction trials using cryopreserved sperm (p < 0.05); 3) cryopreservation did not affect settlement success, as larvae produced with fresh or cryopreserved sperm had the same settlement success (p > 0.05); and 4) the residence time of the sperm within the bank was not important as the fertilization success of sperm frozen for less than 1 month was similar to that frozen up to 2 years (p > 0.05). These results described the first settlement for coral larvae produced from cryopreserved sperm and established important ground-work principles for the use of cryopreserved coral sperm for future reef restoration efforts.

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze-thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid ( 2 40 and 2 100 8C/min) cryopreservation by incubation in HEPES-buffered Ham's F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean6 S.E.M. sperm motility post-thaw (56.1 6 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 6 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 6 1.7% versus cryopreservedthawed, 81.7 6 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 6 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 6 1.1%). Frozen-thawed sperm preincubated without accelerators underwent capacitation (49.6 6 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 6 1.4%) and without accelerators (9 h: 41.2 6 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

For some species, embryos cultured with conspecific companions may have enhanced in vitro development compared with singletons. The objective of this study was to determine the effect of quality and age of companion embryos on single felid embryos produced by in vitro maturation or in vitro fertilization. Test oocytes (intermediate quality) were inseminated and incubated alone or with 10 embryos derived from oocytes with a high, intermediate, or low glucose uptake. The effect of relative age of companion embryos on test embryo development was also examined by insemination and incubation of test oocytes alone or with 10 conspecific embryos that were older, younger, or the same age. Test embryos coincubated with better- or equal-quality companions had better development and more cells per embryo (mean +/- SEM number, 74.9 +/- 16.9 and 40.6 +/- 8.8, respectively, Day 7; P < 0.05) than test embryos coincubated with lesser-quality companions (5.1 +/- 1.4) or alone (8.4 +/- 3.7). Intermediate-quality embryos incubated with older companions had more cells per embryo (88.3 +/- 17.0; P < 0.01) than those incubated with synchronous (49.3 +/- 12.1) or younger (29.4 +/- 6.1) embryos. The cell number of solitary embryos (9.8 +/- 3.1) was less (P < 0.05) than that of every group of test embryos incubated with companions, regardless of age. In vitro development of solitary cat embryos is improved by culture with excellent-quality conspecific companions, particularly companions of an advanced age.

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