Using biological tests to monitor dairy cow fertility, reproductive hormones and other metabolites

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Reproductive problems in dairy cows frequently begin prior to calving. One of the regular practices you should consider when subfertility is suspected in your herd is to use lab tests that may help detect critical physiological changes happening in the cow around calving that otherwise would go unnoticeable until a clinical condition appears. Even worse, when the clinical condition is diagnosed, reproductive failure is already under way.

Important management problems during the transition period or in your regular heat detection program can be monitored with the lab tests presented in this article. The objective of this article is to outline and briefly describe the most common reproductive and metabolic parameters that producers and consultants can use to identify possible problems related to fertility in high producing dairy herds. An important aspect when collecting samples to measure reproductive hormones or metabolites is sample size and time of collection, so management decisions can be made with confidence.


Under normal conditions, circulating progesterone is very low (<1 ng/mL of serum) for cows displaying signs of heat, and will stay low for about three days. Then progesterone begins to rise above 1 ng/mL, reflecting the activity of a new corpus luteum during the remaining of metaestrus and diestrus. Producers can collect blood samples from cows at the time of insemination for either heat detection or fixed-time A.I. (TAI) programs. With this procedure we can estimate the proportion of cows with high progesterone at the time of insemination, thus determining the proportion of false positive heats. Ideally another blood sample should be collected 7 to 10 days after A.I. to detect anovular cows.</p />

Normally, no more than 10% of the cows should have progesterone in serum greater than 1 ng/mL. As pointed out by several research groups, cows with high circulating progesterone near A.I. are less likely to conceive, as shown in Figure 1 below (adapted from Souza et al., 2007).

Progesterone can be measured from the serum or milk samples and is a useful tool to evaluate the proportion of cows not cycling at the end of voluntary waiting period (VWP). And more importantly it can be used to track compliance on a TAI protocol or to determine accuracy of estrus detection as stated above.

Based on this information, your herd veterinarian can create a benchmark or change management practices in order to induce cyclicity and maximize fertility results. In addition, your herd veterinarian can use therapeutic treatments to induce cyclicity for individual animals. You, as a manager, can also review your heat detection program and responsibilities for injections during your TAI protocol.

In research, daily individual progesterone and lutenyzing hormone (LH) measurements are also used to track the profile of these hormones during the estrous cycle. Thus, it is possible to predict the phase of the estrous cycle and the time of ovulation based on intense progesterone and LH samplings. Unfortunately, this technique is somewhat expensive and labor intensive, therefore, not considered for practical application in normal reproductive routines on dairy farms.

The number of cows to be sampled varies with herd size, and insemination protocol, since they will determine the number of cows inseminated in a single day. Ideally a minimum of 30 cows should be sampled.

Get assistance from you herd veterinarian to collect blood samples if needed. Follow the standard protocol for blood collection from the coccygeal artery or vein (tail vein) using a 10 cc vacuum tube without anticoagulants or additives.

Collected blood samples should be stored at upright position (see example in Picture 1) in the refrigerator for one (1) hour to allow for serum separation. Transfer the serum to a smaller sterile container (1.5 or 2.0 ml Ependorf container. Example in Picture 2.) and put all serum samples in the freezer. You can keep collecting and freezing samples until you have enough to justify shipment and analysis, which is about 30 samples. Ship the serum samples with lots of ice in a thermo-isolated container with overnight delivery to Accelerated Genetics headquarters in Baraboo, Wis. or the lab of your choice.


Ketone bodies build up in the bloodstream when the pre-fresh or fresh cow’s liver is over saturated with fat mobilized from body reserves and there is not enough glucose to convert it into energy. In this case, fat takes an alternative pathway and is converted to ketone bodies. It is known that higher circulating ketone bodies near calving are correlated with lower fertility results in dairy cows.

A recent report (Walsh et al., 2007), using a large number of dairy cows, defined a threshold for circulating ketone bodies at two weeks (14 days) postpartum and subsequent pregnancy outcomes. They observed that cows presenting serum BHBA ≥ 1,400 μmol/L (or 14.4 mg/dL) were less likely to conceive. It was also found that animals that did not present high levels of BHBA postpartum had days open equals to 108. In contrast, animals with high levels of BHBA in the first two weeks postpartum had days open equals to 130.

Measurements can be performed in the farm with the use of ketone strips or digital devices. Blood samples should be collected from cows about 5 hours after the last feeding to catch the peak BHBA concentration. For BHBA, a minimum of 12 cows should be sampled. The target group for this test should be cows between 5 and 50 days in milk (DIM).

Thus, consultants and producers can track the percentage of cows with BHBA greater than 1,400 μmol/L. Preferably, no more than 10% of the cows should present high BHBA levels in blood.


Presence of NEFAs in the blood is a direct indicator of massive fat mobilization, which suggests that cow demands for energy are much higher than supplied in the diet; therefore, the NEFA analysis can indicate whether a herd has abnormal levels of negative energy balance.

We should not track mean values, but rather the proportion of cows above a certain threshold level of NEFAs. For cows from 2 to 14 days before calving, the adequate threshold value is 0.400 mEq/l. In other words, we need to calculate the proportion of cows with NEFA levels higher than this threshold in the prepartum. It is recommended that no more than 10% of the cows are above this threshold.

Blood samples for NEFA analysis should be collected just before the feeding time. If a cow calves soon after sample collection (less than 2 days) her sample should be interpreted with caution or removed from analysis. The recommended minimum sample size is 12 cows.


Ruminal pH can be evaluated to measure the risk for sub acute ruminal acidosis (SARA) in the herd. It is being shown that herds with higher proportion of cows with ruminal pH ≤ 5.5 have lower fertility.

Ruminal samples can be collected at five (5) hours after the last feeding, from cows at 5 to 150 DIM, with appropriate needle and syringe as described by Garrett et al., 1999.

No more than 25% of the cows should present ruminal pH ≤ 5.5. f more than 25% of the cows have uminal pH ≤ 5.5, it means that this given herd is going through some risks related with subclinical acidosis and changes in diet should be performed. For SARA a minimum of 12 cows should be sampled.


It is widely known that high levels of MUN are associated with lower fertility in dairy cows. In general, most of the research articles show that if MUN is ≥ 18-19 mg/dL, conception is generally compromised, whereas optimal MUN values for lactating dairy cows are 12-16 mg/dL.

Some diagnosis centers calculate milk urea (MU, g/L) rather than MUN (mg/ dL). Thus, the conversion from MU to MUN can be made simply by multiplying MU by 47, just because in weight basis urea is 47% nitrogen. Sampling time related to feeding should be about 3 hours after a major protein is fed. The minimum recommended sample size is 8 cows at any stage of lactation.

Reproductive success is always a challenge in dairy cattle operations. To learn how to use these diagnostic tools to monitor reproduction in your herd contact your herd veterinarian or your local Accelerated Genetics’ Reproduction Support Team or call 800-451-9275 or e-mail Humberto Rivera at or Alex Souza at


Effect of estradiol-17 supplementation before the last GnRH of the Ovsynch protocol in high producing dairy cows. 2007. A.H. Souza, A. Gümen, E.P.B. Silva, A.P. Cunha, J.N. Guenther, C. M. Peto, D. Z. Caraviello and M.C. Wiltbank. 2007. J. Dairy. Sci. 90:4623-4634.

Rumenacidosis with special emphasis on diagnostic aspectsof subclinical rumen acidosis: a review. 2002. Jörg Matthias Dehn Enemark, Rolf Jess Jørgensen, Peter St. Enemark. Vet. Zoot. 20:1392-2130.

Diagnostic methods for the detection of subacute ruminal acidosis in dairy cows. 1999. E.F. Garrett, M.N. Pereira, K.V. Nordlund, L.E. Armentano, W.J. Goodger, G.R. Oetzel. J. Dairy Sci. 82:1170-1178.

The effect of subclinical ketosis in early lactation on reproductive performance of postpartum dairy cows. 2007. R.B. Walsh, J.S. Walton, D.F. Kelton, S.J. LeBlanc, K.E. Leslie, and T.F. Duffield. J. Dairy. Sci. 90:2788-2796.

Update on Dairy Herd Nutrition Troubleshooting. 2000. Garret Oetzel. Proceedings, 16th Annual Postgraduate Conference - Large Animal Proceedings, School of Veterinary Medicine, University of Wisconsin-Madison. p 109-126.


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