Breeding management 102 - It's all in the timing (Proceedings)

Introduction

Canine breeding management centers hinges on ovulation timing. Ovulation timing is important because it is critical to have viable spermatozoa in the bitch's oviducts at the time the eggs are mature and ready to be fertilized. In many bitches, the period of sexual receptivity (standing and flagging for the male) does not coincide with the bitch's most fertile period. Breeding the bitch based on sexual receptivity, when it does not coincide with the fertile period will result in non-pregnancy; or alternatively if the period of receptivity overlaps somewhat (either at the beginning or at the end) with the fertile period, conception may result but litter size will be reduced. Optimal breeding management is designed to time the breeding precisely during the period of peak fertility. Not only does this result in maximal pregnancy rates and litter size but it also minimizes the number of breedings required, thus reducing the potential for overuse of the dog.

Great advances in canine assisted reproduction techniques have been achieved during the past 20 years. In additional to natural breeding, artificial inseminations with fresh "neat" (unextended) semen, fresh-chilled extended semen and frozen semen are now viable breeding options. With each technological advance, so too come more manipulations by human hands and an increased risk for human error in semen processing and insemination.

When good quality, fresh semen is used for breeding with either natural mating or "side by side" artificial inseminations, there is much forgiveness with respect to breeding timing. This is because fresh semen remains highly viable and "fertilizable" for 5 to 6 days once deposited in utero. Live spermatozoa have been recovered from the uterus up to 11 days post-breeding. In contrast, survival of fresh, "neat" semen in extrauterine environments is poor.

Advances in preserving extrauterine longevity of spermatozoa include both reducing energy consumption and increasing energy sources by and for the sperm cells. Lowering the temperature of sperm cells slows metabolism and reduces their energy consumption. Replacing seminal plasma with extender bathes sperm cells in an energy-rich environment. However, even optimally extended and cooled spermatozoa fail to survive nearly as long as their fresh, intrauterine counterparts unless the extender is replaced with a new aliquot every 48 to 72 hours.

Fresh-chilled sperm cells have a relatively short life span. Fresh-chilled sperm cells, once warmed to body temperature and in utero, lives about 24 to 72 hours. Properly frozen sperm cells can be stored indefinitely in liquid nitrogen at -322°F (-180°C). Once thawed, however, sperm cells have an ultra-short life span. Frozen-thawed spermatozoa live an average of 12 hours, 24 hours maximum! Curtailed sperm cell longevity necessitates precise ovulation timing and breeding.

Ovulation timing

There is an art to the science of ovulation timing (OVT) in the canine. Unlike other species in which ovulation occurs in an estrogen environment, ovulation in the canine occurs only after progesterone levels have risen to >4ng/ml. Another unique feature is that the bitch ovulates ova in the primary oocyte stage. These primary oocytes must undergo another meiotic division and further maturation before they become secondary oocytes and can be fertilized. Unlike most species in which breeding/insemination is timed to coincide with ovulation, insemination in the bitch is performed 2 to 4 days after ovulation. It is important to remember that, in the bitch, the critical timing events occur 4 to 6 days before the optimal breeding dates.

A number of tools are available for ovulation timing. These include LH (luteinizing hormone) assays, qualitative and quantitative progesterone assays, vaginal epithelial cytology, vaginoscopy, examination of external genetalia and behavioral assessment. Success in breeding is optimized when as many tools as possible are utilized to time ovulation.

Estrogen

Increased estrogen causes an increased turnover rate of vaginal epithelial cells, resulting in the progressive cornification seen on vaginal cytology, and thickening of the vaginal wall in preparation for breeding. Also seen is progressive edema of the vaginal mucosa, which can be visualized with endoscopic examination. Estrogen assays are performed by radioimmunoassay (RIA) by many commercial laboratories. However, the information given is of little value for ovulation timing, since peak estrogen levels are variable from bitch to bitch, and even relative changes do not correlate to ovulation or the fertile period. Estrogen is best assessed by serial vaginal cytology evaluation and vaginoscopy. Estrogen levels do not indicate the fertile period since ovulation is triggered by the LH surge, not an estrogen peak.

Estrogen levels rise, peak and begin to decline during proestrus. Under the influence of estrogen, the vulva and vagina become edematous, a serosanguinous vulvar discharge (of uterine origin) is present, the vaginal epithelium becomes thickened and cornified and the bitch exhibits behavioral signs of estrus. However, blood estrogen levels are highly variable, do not accurately predict ovulation and are not useful as a tool for timing ovulation.

LH (luteinizing hormone)

Hypophyseal LH is the biological trigger for the cascade of events that culminate in ovulation. In both sexes, LH stimulates secretion of sex steroids from the gonads. In males, LH binds to receptors on Leydig cells in the testis, stimulating synthesis and secretion of testosterone. In females,theca cells in the ovary respond to LH stimulation by secretion of testosterone, which is converted into estrogen by adjacent granulosa cells.

Following a variable period (1 to 21 days) of estrogen level elevation, the LH surge signals the transition from proestrus to estrus. LH stimulates follicular granulosa cells to secrete progesterone. The LH surge is the key timing event dictating days of peak fertility. Ovulation begins 2 days post-LH surge and continues for another day or so. Ova mature and are capable of fertilization 2 days later. Mature ova live another 1-3 days. Therefore, optimal breeding times are day 4, 5, and 6 post-LH surge.

Pinpointing the day of the LH surge is cumbersome. The LH surge is short-lived, typically lasting only 12 to 24 hours. Therefore, daily blood testing is required. LH testing is available through commercial laboratories or as an in-house snap test. A qualitative (test kit) assay [Witness LH, Synbiotics Corp.] is available for in-hospital use, but it has a short shelf life (3 months maximum). In many cases, the LH surge can be identified retrospectively, once progesterone reaches 2 ng/ml (6.2 nmol/l), From a practical standpoint, daily serum samples are saved and tested retrospectively once the serum progesterone reaches 2 ng/ml. LH assays are most often used in the timing for breedings with frozen semen. If a single insemination is to be performed, the optimal day for breeding is day 5 after the LH surge (LH surge = day 0).

Progesterone

Progesterone's initial rise occurs concomitantly with the LH surge. At that time, baseline progesterone levels (<1.5ng/ml) rise to 1.5-2.0 ng/ml. After the initial rise, progesterone continues to rise and may reach levels of 10-15 ng/ml by the end of the fertile period. Ovulation occurs when progesterone levels are between 4 and 10 ng/ml. Plan breedings 4 to 6 days after the initial rise, and 2 to 4 days after the onset of ovulation. Since baseline and initial rise levels can vary from individual to individual, it is important to start testing early enough to define the baseline progesterone level.

The gold standard for determining progesterone levels is quantitative measurement by radioimmunoassay (RIA). RIA's must be performed in a laboratory approved for the use of radioactive isotopes, aka "hot labs." Some of the newer chemiluminescent assay machines (eg. Immulite by Siemens healthcare Diagnostics, Inc.) offer an accurate in-house means of progesterone testing. Results of quantitative tests are reported in ng/ml. Progesterone, like all steroid hormones, is not species-specific and can be measured commercially by human and veterinary laboratories alike.

Several semiquantitative ELISA progesterone kits are available, Status-Pro [Synbiotics], Target [Biometallics], and PreMate [Camelot Farms], for example. Results are interpreted from a color change in the test well or membrane. Hemolyzed blood samples will falsely lower the result. If test kit components are not warmed to room temperature, then a falsely high result will be interpreted. The general consensus is that test kits are usually adequate for most natural breedings, but are not accurate enough when planning a fresh chilled or frozen breeding.

Recommendations for progesterone testing include starting to test around day 5 or 6 from onset of sign of proestrus, followed by testing every other day until ovulation has been confirmed. However, some females have very short seasons and require testing at the first sign of proestrus. Others may not ovulate until after 21 or more days. When in doubt, start testing early.

Following ovulation it takes the eggs 2 days to mature and be ready to be fertilized. Ovulation of multiple follicles may occur over a 12 – 36 hour period. Once mature, the eggs are capable of being fertilized for 2 days. Therefore, the fertile period begins 2 days after ovulation and lasts for the next 3 – 4 days. It is recommended that ovulation be confirmed (rather than presuming a normal progression of ovulatory events after the progesterone surge) because of the tendency for some bitches to stall between the surge and ovulation or for some bitches to fail to ovulate completely (due to split heats, inadequate hormone production from the brain, or abnormal follicular development).

Typically, the first day of breeding (day 4 after the LH surge, day 2 after ovulation) will occur when the progesterone concentration is between 12 and 15 ng/ml (37.2 and 46.8 nmol/l). Progesterone concentrations rise to between 4 and 10 ng/ml at the time of ovulation (12.6 and 31.4 nmol/l). Before ovulation, progesterone rises slowly, while after ovulation progesterone rises rapidly. A jump of at least 3 – 4 ng/ml (9.14– 12.56 nmol/l) in a 24 hour period, after progesterone concentrations reach 4 ng/ml, is confirmatory of ovulation. Some bitches may jump as much as 8 – 10 ng/ml in a 24 hour period after ovulation has occurred. Some bitches will stall between the progesterone surge and ovulation having values between 2.5 and 4.0 ng/ml (7.8 and 12.5 nmol/l) for several days.

Evaluation of external genetalia

During proestrus when the tissues are under the influence of estrogen, the vulva is swollen and a sersanguinous vulvar diacharge (of uterine origin) is present. As proestrus transitions to estrus, vulvar edema diminishes and the vulvar discharge becomes straw colored. Note however, that some normal bitches may have a hemorrhagic discharge that persists throughout estrus. With the onset of diestrus, vulvar edema subsides completely.

Behavior

The period of receptivity to a male varies considerably among bitches; some bitches are receptive well before and after the period of potential fertility. The proestrous bitch attracts males but will not stand to be mounted. During estrus, most bitches will stand, "flag" and allow the dog the mount and intromit. There are exceptions. Bitches of high social status may not allow themselves to be mounted even during the period of peak receptivity. With the onset of diestrus, the bitch will refuse to stand and be mounted.

Exfoliative vaginal cytology

Examination of the cells on the surface of the vaginal epithelium will give information about the stage of the estrous cycle. Proper technique is important so that the cells obtained are representative of the hormonal changes occurring. The sample should be collected from the cranial vagina, since cells from the clitoral fossa, vestibule and/or caudal vagina are not as indicative of the stage of the cycle. Since cytologic changes reflect the underlying endocrine events of the cycle, they are almost always a better predictor of the "fertile time" and gestation length than are behavioral or physical signs.

The vaginal epithelium is responsive to sex steroids, particularly estrogen, and undergoes predictable changes through the cycle in response to changes in blood concentrations of ovarian hormones. In essence, vaginal cytology is a type of endocrine assay. Tracking changes in the morphology of desquamated vaginal epithelial cells provides a convenient means of assaying changes in estrogen levels. Examination of a single smear can sometimes provide useful information, but can also be quite misleading. For example, it is often difficult to differentiate proestrus and diestrus from an isolated smear. It is therefore highly recommended that multiple smears be evaluated.

Under the influence of rising estrogen levels, the number of layers making up the vaginal epithelium increases dramatically, presumably to provide protection to the mucosa during copulation. Rising levels of estrogen also cause the vaginal epithelium to become "cornified" - the surface cells become large and flattened, with small or absent nuclei. Therefore, as estrogen rises during proestrus, the maturation rate of the epithelial cells increases, as does the number of keratinized, cornified epithelial cells seen on a vaginal smear.

During early proestrus, estrogen concentrations are low, and cytology specimens show parabasal and intermediate cells, red blood cells, neutrophils and bacteria. As proestrus progresses, estrogen levels rises and the vaginal epithelium thickens. The number of superficial cells increases and the number of red blood cells gradually decreases. By the end of proestrus, more than 80% of the epithelial cells are cornified red blood cells.

During estrus, vaginal smears contain >90% cornified superficial epithelial cells, red blood cells are few in number and neutrophils are absent. Full cornification continues throughout estrus, until the "diestral shift" that occurs 7 to 10 days after the LH surge, signifying the first day of diestrus. The vaginal smear then changes abruptly from full cornification to 40-60% immature (parabasal and intermediate) cells over a 24-36 hour period. If vaginal cytology is performed until the diestral shift is observed, a retrospective analysis of the LH surge, ovulation and the fertile period can be obtained.

With the onset of diestrus, vaginal cytology abruptly changes from that seen during estrus to one showing few superficial cells, a predominance of parabasal and intermediate cells and many neutrophils.

Practically, vaginal cytology samples are useful for identifying inflammation in the reproductive tract. If a large number of neutrophils are seen on an estrus smear, then a vaginal culture and sensitivity may be indicated. Cytology is also useful for differentiating proestrus, estrus and diestrus. It is not useful for prospective determination of ovulation date within the estrous period. However, determining the onset of cytologic diestrus allows a retrospective prediction of optimal breeding times and a prospective prediction of whelping date. Anticipated whelping date can accurately be calculated to be 56 to 58 days from the onset of cytologic diestrus.

Vaginoscopy

Vaginoscopy is a useful tool for estimating the LH surge and optimally fertile period. Just as the cell types change as the bitch progresses through her cycle, so does the appearance of the vaginal epithelium. During proestrus, the walls of the vagina are uniform pink in color and edematous (puffy or billowy) on vaginoscopy. Once estrogen levels begin to decrease (late proestrus/early estrus) the vaginal wall begins to dehydrate and the blood supply to the vaginal wall diminishes. This results in the vaginal wall becoming pale pink and finally white in color and in wrinkling of the tissues (crenulation) as the fluid is reabsorbed. This usually coincides with the time of the LH surge. For the remainder of estrus, the vaginal wall appears white and wrinkled (crenulated). Maximal crenulation occurs during the optimal fertile period several days later. Once the bitch enters diestrus (past the fertile period), the vaginal wall regains its pink color. However, the color is not uniform, but rather blotchy pink and red. The mucosa is very irritable and if it is touched with an instrument or swab it will pucker up (rosette formation).

Review of canine reproductive physiology/endocrinology

     • Oocytes are ovulated into a progesterone, not estrogen, environment.

     • Oocytes are ovulated as primary oocytes and are not yet ready to be fertilized.

     • Optimally-timed breeding/insemination does not coincide with ovulation.

     • Critical timing events occur 4 to 6 days before the optimal breeding dates.

     • Optimally, insemination occurs 2 to 4 days post-ovulation.

     • Fresh semen remains highly viable and "fertilizable" for 5 to 6 days once deposited in utero. Live spermatozoa have been recovered from the uterus up to 11 days post-breeding.

     • Fresh-chilled spermatozoa, once warmed to body temperature and in utero, live about 24 to 72 hours.

     • Properly frozen sperm cells can be stored indefinitely in liquid nitrogen at -322°F (-180°C).

     • Once frozen then thawed, spermatozoa live an average of 12 hours, 24 hours maximum!

Ovulation timing

Always pinpoint ovulation date using all available tools.

     • Exfoliative Vaginal Cytology

          - Serial evaluation of samples obtained on a daily basis are most useful

          - Can confirm the presence or absence of inflammation in the vagina

          - Can differentiate between proestrus, estrus and diestrus

          - Cannot prospectively pinpoint ovulation date

          - Can prospectively predict whelping date

     • LH (Luteinizing Hormone)

          - Hypophyseal LH is the biological trigger for the cascade of events that culminate in ovulation.

          - Optimal breeding times are day 4, 5, and 6 post-LH surge.

          - The LH surge is short-lived, typically lasting only 12 to 24 hours. Therefore, daily blood testing is required.

          - In-house test kits available [Synbiotics Corporation, San Diego, CA], short shelf life (3 months maximum). LH assays are most often used to time frozen semen breedings.

     • Progesterone

          - Send blood out for an RIA.

          - Start testing on day 5, 6, or 7 from the onset of proestrus.

          - Test every 2nd or 3rd day until ovulation is confirmed.

          - Baseline progesterone levels are <1.5ng/ml.

          - An initial rise occurs concomitantly with the LH surge (1.5-2.0 ng/ml).

          - Ovulation occurs when progesterone levels reach 5 to 6 ng/ml.

          - Optimal breeding times are day 2, 3, and 4 post ovulation

     • Estrogen

          - Responsible for vulvovaginal edema, the serosanguinous vulvar discharge, and vaginal epithelial cornification

          - Levels are highly variable, do not accurately predict ovulation and are not useful as a tool for timing ovulation.

     • Vaginoscopy

          - Proestrus - vaginal mucosa edematous ("billowing pillow")

          - LH Surge - vaginal edema decreases, vaginal folds crenulate

          - Optimal fertile period – maximal crenulation

          - Diestrus approaching - vaginal mucosa blotchy white and pink

     • Evaluation of External Genetalia

          - Proestrus - vulva is edematous; serosanguinous discharge present

          - Estrus - vulvar edema diminishes; discharge becomes straw colored

          - Diestrus - vulvar edema subsides completely; no discharge

     • Behavior

          - Proestrus - bitch attracts males but will not stand to be mounted.

          - Estrus, the bitch will stand, "flag," allow the dog the mount and intromit.

          - Diestrus, the bitch will refuse to stand and be mounted.

Key points

     • Start ovulation timing early (day 5, 6 or 7 from first sign of proestrus), particularly in females who have not had their cycles "tracked" before.

     • Use all available tools for ovulation timing (exfoliative vaginal cytology, hormonal assays, vaginoscopy, examination of external genetalia, and behavior).

     • Blood sampling for LH assays must be performed daily.

     • Blood sampling for progesterone levels is usually performed every other day.

     • Don't use qualitative or semi-quantitative assays for progesterone.

     • Do use RIA (radioimmunoassay) progesterone level determination.

     • Determine the bitch's date of ovulation (2 days post LH surge and progesterone 5-6 ng/ml).

     • Confirm that ovulation occurred.

     • Natural matings and nonsurgical A.I.'s are scheduled 2 and 4 days post ovulation.

     • Surgical inseminations are performed 3 days after ovulation. (E.g. If ovulation occurs on February 11th , then the surgery is performed on February 14th.)

     • Ovulation date, not breeding date(s), is the most accurate predictor of parturition date.

     • Parturition occurs 63 days ± 24 hrs after ovulation.

References/suggested reading

Barber JA. It's all in the timing –Fresh Chilled vs. Frozen Semen Breeding in the Canine. Proc An Meeting Soc Therio. Lexington, KY: 2004;253-263.

Davidson A, Baker T. Controversies in ovulation timing (proceedings), CVC Proc, Apr, 2009.

Edens MSD, Heath AM. Breeding management in the bitch and queen. In: Root Kustritz MV, ed. Small animal theriogenology.St. Louis: Elsevier, 2003;33-60.

Gotwals S. Canine reproduction seminar. Proc AKC Canine Health Foundation Seminar, University of Florida, March 2000, 1-33.

Johnston SD, Root Kustritz MV, Olson PNS., Breeding Management and Artificial Insemination in the Bitch. In: Canine and feline theriogenology, Philadelphia: WB Saunders Co, 2001;105-128.

Van Hutchison R. Maximizing conception rate. In: Canine Reproduction for Breeders, The Iams Company, Dayton, OH, 2001; 8-12.

Linde-Forsberg C. Fertility data from 2041 controlled artificial inseminations in the dog. In: Proc 4th Internat Symp on Canine and Feline Reprod, Oslo, 2000; 120.

Linde-Forsberg C, Ström Holst B and Govette G. Comparison of fertility data from vaginal vs intrauterine insemination of frozen thawed dog semen: A retrospective study. Theriogenology 1999; 52:11-23.

O'Connor C, Traas AM. Dvanced canine reproductive techniques:the most current approaches in breeding management.J Vet Behav 2009, 4(6):2560257.

Thomassen R, Farstad W, Krogenaes A, Fougner JA and Andersen Berg K. Artificial insemination with frozen semen in the dog. A retrospective study. J Reprod Fertil Suppl 2001; 57:341-346.

Wilson M. Non-surgical intrauterine artificial insemination in bitches using frozen semen. J Reprod Fertil Suppl 1993; 47 (Suppl):307-311.