CLONING AND STEM CELLS Volume 10, Number 3, 2008 © Mary Ann Liebert, Inc. DOI: 10.1089/clo.2008.0001 Protocol for the Recovery of In Vivo Matured Canine Oocytes Based on Once Daily Measurement of Serum Progesterone Mohammad Shamim Hossein, 1 Yeon Woo Jeong, 1 Sue Kim, 1 Jeong Joo Kim, 1 Sun Woo Park, 1 Chang Soo Jeong, 1 Sang Hwan Hyun, 1,2 and Woo Suk Hwang 1 Abstract The collection of in vivo matured canine oocytes relies on the accurate prediction of ovulation. The present study was designed to develop a protocol for the recovery of in vivo matured canine oocytes based on once daily measurements of serum progesterone (P 4 ) concentrations. Blood samples (2 mL) were collected every day at 0900 h, and P 4 concentrations were analyzed using a DSL-3900 ACTIVE ® Progesterone Coated-Tube Radioim- munoassay Kit. The average number of oocytes at the metaphase II (M II) stage was significantly higher at or after 72 h (6.7 to 7.5) compared to 56 h (1.7) following ovulation. The highest numbers of corpora lutea, and therefore the highest numbers of oocytes, were recovered from bitches with initial ovulatory P 4 concentrations ranging from 6.0 to 8.0 ng/ mL (12.2 and 11.4, respectively) compared to from 4.0 to 4.9 ng/ mL (9.6 and 8.8, respectively; p  0.05). The average number of M II oocytes recovered at 84 h from bitches with initial ovula- tory P 4 levels of 5.0 to 5.9 ng/mL (7.7) was higher compared to bitches with P 4 levels of 4.0 to 4.9 ng/ mL (3.5) and 6.0 to 8.0 ng/ mL (4.8; p  0.05). When oocyte recovery time was adjusted for initial ovulatory P 4 concen- tration, no significant difference in recovery rates or oocyte quality were observed. In conclusion, once daily measurements of P 4 can be used to predict ovulation in bitches, and oocyte recovery time should be adjusted for initial ovulatory serum P 4 concentrations. 403 Introduction A THOROUGH UNDERSTANDING of reproductive physiology is essential when designing a protocol for any oocyte recovery program. The reproductive physiology of bitches is unique in nature. Ovulation may occur within a wide range of times. Generally, between 2 days before and 7 days after the initiation of behavioral estrus, most frequently between 1 and 3 days after the initiation of estrus (Wildt et al., 1978). Bitches ovulate immature oocytes at prophase of the first meiotic division, and oocyte maturation continues in the dis- tal part of the oviduct for at least 48 to 72 h (Holst and Phemister, 1971). In most cases, no oocytes can be found at the M II stage earlier than 56 h postovulation and oocytes start to degenerate after 96 h (Hossein et al., 2007; Mondain- Monval et al., 1993; Reynaud et al., 2005). Therefore, an early or late attempt at oocyte collection may lead to the recovery of immature, aged, or degenerated oocytes. Indeed, in our earlier study, numerous immature and aged oocytes (30%) were recovered by flushing the fallopian tubes (Hossein et al., 2007). Thus, successful collection of matured oocytes de- pends on an accurate prediction of ovulation and the deter- mination of an appropriate time for oocyte recovery in rela- tion to ovulation. Various methods including vaginal cytology, the degree of vulval tumescence, and vaginoscospic examination can be used to predict reproductive status in bitches (Goodman, 2002). Although these methods give an indication of ovula- tion, their repeatability and accuracy is highly variable. Ad- ditionally, a real-time diagnostic B-mode ultrasound can monitor follicular growth and detect ovulation (England and Allen, 1989). Some studies have found this method to be very convincing (Hase et al., 2000); however, ovulation is difficult to identify because canine follicles do not collapse after ovu- lation, echogenic changes are not always consistent, and new corpora lutea have central fluid filled cavities similar to those of preovulatory follicles (Boyd et al., 1993). The measurement of reproductive hormones, specifically luteinizing hormone 1 Sooam Biotech Research Foundation, Seoul, Republic of Korea. 2 Laboratory of Veterinary Biotechnology, College of Veterinary Medicine, Chungbuk National University, Chungbuk, Republic of Ko- rea.